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Striatal opioid peptide content in an animal model of spontaneous stereotypic behavior
Behavioural Brain Research 157 (2005) 363–368
Research report
Striatal opioid peptide content in an animal model of spontaneous stereotypic behavior Michael F. Presti∗ , Mark H. Lewis1 Departments of Psychiatry and Neuroscience, University of Florida, 100 S. Newell Dr., P.O. Box 100256, Gainesville, FL 32610, USA Received 27 April 2004; received in revised form 23 July 2004; accepted 5 August 2004 Available online 19 September 2004
Abstract Stereotypic behaviors are associated with a broad spectrum of developmental, psychiatric, and genetic disorders. Several lines of evidence indicate that these abnormal repetitive behaviors may be expressed as a consequence of imbalanced activity along the direct and indirect processing pathways of the basal ganglia. Because the activity of these two pathways is modulated, in part, by the neuropeptides dynorphin and enkephalin, we sought to determine whether spontaneous and persistent stereotypy is associated with alterations in striatal neuropeptide content. Specifically, the present study employed radioimmunoassay to measure dynorphin-A and leu-enkephalin content in the dorsolateral striatum of deer mice exhibiting different levels of spontaneous stereotypic jumping. The results indicate significantly decreased leu-enkephalin content and significantly increased dynorphin/enkephalin content ratios in high-stereotypy mice relative to low-stereotypy mice. Moreover, analyses revealed a significant negative correlation between striatal enkephalin content and frequency of stereotypy as well as a significant positive correlation between the dynorphin/enkephalin content ratio and frequency of stereotypy in these mice. These data are consistent with the hypothesis that spontaneous stereotypic behavior is expressed as a consequence of relative hyperactivity along cortico-basal gangliacortical feedback circuits involving the direct (facilitative) pathway, but suggest that primary perturbations to the indirect (inhibitory) pathway give rise to such imbalanced activity. © 2004 Elsevier B.V. All rights reserved. Keywords: Stereotypy; Dynorphin; Enkephalin; Striatum; Direct pathway; Indirect pathway
1. Introduction Several lines of evidence have implicated basal ganglia nuclei in the mediation of abnormal repetitive behavior. Pharmacological studies have consistently shown that manipulations which enhance activity of the direct (striatonigral) pathway or which inhibit activity of the indirect (striatopallidal) pathway induce motor stereotypy [5,19,3,17]. Conversely, spontaneously emitted stereotypies are attenuated via pharmacological manipulations which inhibit activity of the direct pathway [14] or which enhance activity of the indirect pathway [15]. Moreover, the intense stereotypy induced by ∗
Corresponding author. Tel.: +1 352 294 0427; fax: +1 352 294 0425. E-mail addresses: [email protected] (M.F. Presti), [email protected] (M.H. Lewis). 1 Tel.: +1 352 294 0415; fax: +1 352 294 0425. 0166-4328/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2004.08.003
psychostimulant drugs is associated with increased expression of immediate early genes in striatonigral neurons [2]. Interestingly, transgenic animals that selectively overexpress FosB (an immediate early gene involved in the modulation of neuronal excitability) in striatonigral projection neurons exhibit perseverative wheel running, whereas wheel running is significantly inhibited in animals that overexpress the gene in striatopallidal projection neurons [23]. These studies support the hypothesis that stereotypic and perseverative behaviors are expressed as a result of imbalanced activity along the direct and indirect pathways of the basal ganglia and that this imbalance is characterized by a relative increase in striatonigral tone. Output of the striatonigral and striatopallidal pathways is modulated in part by the neuropeptides dynorphin and enkephalin, respectively, which are specifically localized within the striatal medium spiny neurons comprising these
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pathways (for review, see [21]). Furthermore, studies have shown that the expression level of both peptides is positively correlated with activation of their respective basal ganglia pathways [22,24,11]. Given such modulatory interactions, it is reasonable to assume that perturbations to the normal balance of activity along the two main pathways of the basal ganglia will be associated with alterations in neuropeptide content. Specifically, the dynorphin/enkephalin content ratio is expected to correlate positively with increased relative striatonigral tone and negatively with increased relative striatopallidal tone. Numerous studies provide indirect support for an association between the expression of stereotypic behavior and perturbations to endogenous opioid peptide systems. For instance, apomorphine and amphetamine-induced stereotypies are attenuated via intrastriatal administration of both leu- and met-enkephalin [9,12,1]. Furthermore, it has been reported that mu- and kappa-receptor densities are inversely correlated with stereotypy duration in tethered sows [25]. Similarly, human patients exhibiting repetitive self-injurious behavior demonstrate hypersensitivity to opiate administration [6], and such behaviors are attenuated clinically following administration of the mu opioid receptor antagonist naltrexone [18]. Given the above findings, the present study sought to determine if levels of spontaneous stereotypic jumping are correlated with striatal neuropeptide content in the deer mouse model of abnormal repetitive behavior. Specifically, we used radioimmunoassay to measure the concentrations of leuenkephalin and dynorphin-A in the dorsolateral striatum of phenotypically distinct (high stereotypy (HS) and low stereotypy (LS)) mice. Because previous work has shown that this form of stereotypy is dependent on D1 receptor-mediated striatonigral pathway activation [14], we hypothesized a direct relationship between the dynorphin/enkephalin content ratio and the intensity of stereotypy in these animals.
2. Methods 2.1. Animals The study was performed using 34 adult (>2-month-old) male deer mice (P. maniculatus). As previously reported [13], these mice develop high rates of persistent, spontaneously emitted stereotypy which persist at stable levels when housed under standard laboratory conditions. Mice were group caged (three to four same sex mice per cage) in a standard (29 cm × 18 cm × 13 cm) laboratory mouse cage. Rodent chow and water were available ad libitum, and temperature was maintained at 24 ◦ C. Mice were maintained on a 15/9-h light/dark cycle, with lights off at 10:00 h. All procedures were performed in accordance with the guidelines set forth in the NIH Guide for the Care and Use of Laboratory Animals and were approved by the University of Florida Institutional Animal Care and Use Committee.
2.2. Behavioral testing Mice were individually housed in custom plexiglass testing cages (22 cm × 15 cm × 28 cm) and allowed to habituate to the testing environment overnight (20:00–10:00 h). In order to assess behavioral phenotype, mice were continuously monitored with automated activity monitors (Digiscan) for two consecutive 9-h (10:00–19:00 h) active cycles while housed in the custom testing cages. These cages were designed with slotted plexiglass lids to eliminate cagetop activity-related (e.g., feeding) photobeam interruptions. Water was delivered through a small hole on one wall using a custom-fitted spout, and food was placed inside the cage. Both locomotion and vertical stereotypy (jumping or somersaulting) were recorded throughout the testing period and the data were reported in 1-h intervals. Food and water remained available ad libitum throughout testing. After the second day of testing, mice were returned to their home cages and remained there for 1 week prior to being sacrificed for tissue collection. 2.3. Neuropeptide analyses Animals were sacrificed via cervical dislocation 7 days after behavioral testing. Brains were rapidly removed, snapfrozen in isopentane, and stored at −80 ◦ C. An HM 505E cryostat (Microm) was used to remove a 300 m section of the anterior striatum, which was transferred to a freezing stage (FTS Systems) for micropunch dissection of the anterior dorsolateral striatum. This region was selected for analysis because it corresponds with coordinates used for intracerebral drug administration resulting in selective attenuation of spontaneous stereotypy. Thus, the center of the micropunch was targeted to the following stereotaxic coordinates: anterior to bregma 0.5 mm; lateral 2.5 mm; ventral −2.7 mm. Dissected striatal tissue was sonicated in 100 l of Tris extraction buffer consisting of: 20 mM Tris, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 10 M DTT, 1% Triton X-100, and a protease inhibitor cocktail tablet (Roche). Sonicated samples were placed on dry ice and vortexed every 10–15 min for 1 h, and then centrifuged at 13,200 rpm for 15 min at 4 ◦ C. Supernatant was collected and total protein content was determined in duplicate for each sample using a Thermomax microplate reader (Molecular Devices) and bovine serum albumin as the standard. Total protein content was normalized for each sample, and then leu-enkephalin and dynorphin-A concentrations were determined via radioimmunological assay using commercially available kits (Phoenix Peptide) and the protocols provided by the manufacturer. The antibodies supplied with these kits were raised against synthetic peptides and displayed no significant cross-reactivity with other endogenous opioid peptides. 2.4. Data analysis The experimental data were analyzed in two ways. First, groups of low stereotypy and high stereotypy mice (n = 12 and
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n = 14, respectively) were generated on the basis of average hourly stereotypic frequency over the 18-h course of behavioral testing. Mice were classified as LS if they exhibited an average of less than 300 jumps per hour, and HS if they exhibited an average of more than 1000 vertical beam interruptions per hour. These criteria were designed to be as stringent as possible while maintaining adequate and approximately equal group sizes. In order to evaluate the hypothesized differences in neuropeptide concentrations between these behaviorally distinct phenotypes, t-tests were performed to compare leu-enkephalin concentrations, dynorphin-A concentrations, and leu-enkephalin/dynorphin-A ratios in the LS and HS groups. Next, Pearson product moment correlation coefficients were calculated in order to determine the extent to which dynorphin, enkephalin, and dynorphin/enkephalin levels correlated with stereotypic intensity and locomotor ac-
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tivity using all 34 mice. A p-value of 0.05 was adopted as the threshold for assigning statistical significance on all tests.
3. Results 3.1. Relationship between striatal opioid peptides and stereotypy Statistically significant differences were found in the concentrations of striatal neuropeptides associated with LS and HS mice. Specifically, leu-enkephalin content was significantly lower (t = 2.27, d.f. = 18, p = 0.036) and the dynorphinA/leu-enkephalin content ratio was significantly higher (t = 2.781, d.f. = 18, p = 0.012) in HS mice than in LS mice (see Fig. 1, panels a and c). Dynorphin-A concentrations were
Fig. 1. Phenotypic differences in striatal opioid peptide content. (a) LS mice had significantly greater striatal concentrations of leu-enkephalin than HS mice; (b) no significant differences were detected in striatal dynorphin-A concentrations between HS and LS mice; (c) HS mice demonstrated significantly higher dynorphin/enkephalin content ratios than LS mice.
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Fig. 2. Correlation between striatal opioid peptide content and stereotypic frequency. (a) A significant inverse correlation was detected between striatal leuenkephalin concentrations and stereotypic frequency; (b) no relationship was detected between striatal dynorphin-A concentrations and stereotypy level; (c) a significant positive correlation was observed between dynorphin/enkephalin content ratios and stereotypic frequency.
slightly but not significantly (p > 0.05) higher in HS mice as compared to LS mice (see Fig. 1, panel b). The relationship between opioid concentrations and stereotypy was also assessed using all 34 mice. A significant inverse correlation between leu-enkephalin content and stereotypy level was found (r = −0.404, p = 0.036), as well as a significant positive correlation between dynorphin-A/leuenkephalin content ratios and stereotypy level (r = 0.424, p = 0.027). No correlation (r = 0.007, p > 0.05) was detected between dynorphin content and stereotypy level (Fig. 2, panels a through c).
cant (p > 0.05) elevation was found in the enkephalin content of the low locomotion group, and dynorphin/enkephalin content ratios were slightly but not significantly (p > 0.05) higher in the high locomotion group. Similarly, no significant correlations (p > 0.05) were detected between dynorphin concentrations, enkephalin concentrations, or dynorphin/enkephalin content ratios and locomotor activity (r = −0.05, 0.14, and −0.09, respectively).
3.2. Relationship between striatal opioid peptides and locomotion
Data from the present study indicate that the expression of persistent, spontaneously emitted stereotypy in deer mice is associated with alterations of striatal opioid concentrations and, presumably, the balance of activity along basal ganglia output pathways. Specifically, the inverse correlation detected between the intensity of stereotypy (as reflected by frequency) and striatal enkephalin content suggests that stereotypy is exhibited in these mice as a consequence of abnormal hypoactivity of the striatopallidal pathway. Given the sig-
No relationships were detected between dynorphin-A and leu-enkephalin concentrations or dynorphin/enkephalin content ratios and locomotor activity (data not shown). Specifically, no differences were found in dynorphin content between the 13 highest and 13 lowest activity mice with respect to locomotion (p > 0.05), a small but non-statistically signifi-
4. Discussion
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nificant positive correlation between dynorphin/enkephalin content ratios and stereotypy level, these results are also consistent with the interpretation that the chronic expression of stereotypic behavior is associated with relative hyperactivity of the striatonigral pathway. Thus, the data support the hypothesized stereotypy-related imbalance in activity levels of the direct and indirect pathways. Based on current models of general basal ganglia function, the putative role of the direct striatonigral pathway is to facilitate and sustain the expression of cortically initiated motor programs, whereas that of the indirect pathway is to inhibit and terminate, respectively, competing and completed motor programs [10,8,7]. Given these functional roles, it has been suggested that the pathogenesis of both hypokinetic (e.g., Parkinson’s disease) and hyperkinetic (e.g., Huntington’s disease) movement disorders is linked to specific disturbances of these feedback pathways [4]. We hypothesized that stereotypy, like other hyperkinetic disorders, is expressed as a consequence of imbalanced basal ganglia activity characterized by excessive relative striatonigral tone because such an imbalance would serve as a substrate for sustained activation of selected corticospinal motor programs. This hypothesis is supported by the present findings, which suggest that stereotypy becomes increasingly perseverative with increased relative striatonigral tone. These findings are also consistent with reports that wheel-running is significantly more persistent in transgenic mice which selectively overexpress FosB in striatonigral neurons [23]. There is considerable evidence to support the conclusion that the detected perturbations to peptide content reflect alterations to the balance of activity along basal ganglia output pathways. For instance, intrastriatal administration of the selective D1 dopamine receptor agonist SKF-38393, a manipulation that activates the direct pathway, increases local dynorphin release [24] and expression [20]. Similarly, intrastriatal D2-receptor dopamine antagonist administration, a manipulation that activates the indirect pathway, increases enkephalin protein [22] and mRNA expression [11]. These studies demonstrate that intrastriatal dynorphin and enkephalin concentrations are directly related to the activity levels of the striatonigral and striatopallidal pathways, respectively, and suggest that they can be treated as indices of the relative balance of activity along these pathways. Taken together, the present findings support the hypothesis that stereotypic behavior is expressed as a consequence of excessive activity along cortico-basal ganglia-cortical motor feedback circuits involving the striatonigral pathway. Although the differences in neuropeptide content ratios were expected to be driven largely by increased dynorphin expression, the imbalances were primarily attributable to decreased enkephalin expression. Given the putative role of the enkephalin-modulated indirect pathway in terminating direct pathway-sustained motor activity, this finding suggests that behaviors become repetitive and perseverative due to a loss of indirect pathway-mediated inhibitory control. Interestingly, this is consistent with a previous experiment that pointed to
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impaired indirect pathway function in which the deer mice exhibited intense hyperactivity in response to intrastriatal administration of the D2 antagonist raclopride, a drug which reliably induces catalepsy in other rodents [15]. Therefore, we have interpreted the present results to reflect the fact that impaired function of the striatopallidal pathway in deer mice is associated with a relative increase in striatonigral tone and, accordingly, abnormal repetitive and perseverative behavior.
Acknowledgements We are very thankful to Dr. Violetta Zujovic for her expert assistance with protein extraction. This work was also supported in part by an NRSA fellowship to M.F.P. (DA15357).
References [1] Blumstein LK, Crawley JN, Davis LG, Baldino F. Neuropeptide modulation of apomorphine-induced stereotyped behavior. Brain Res 1987;404:293–300. [2] Canales JJ, Graybiel AM. Patterns of gene expression and behavior induced by chronic dopamine treatments. Ann Neurol 2000;47:S53–9. [3] Chen JF, Beilstein M, Xu YH, Turner TJ, Moratalla R, Standaert DG, et al. Selective attenuation of psychostimulant-induced behavioral responses in mice lacking A(2A) adenosine receptors. Neuroscience 2000;97:195–204. [4] DeLong MR. Primate models of movement disorders of basal ganglia origin. Trends Neurosci 1990;13:281–5. [5] Ernst AM, Smelik PG. Site of action of dopamine and apomorphine on compulsive gnawing behavior in rats. Experientia 1966;22:837–8. [6] Frecska E, Arato M. Opiate sensitivity test in patients with stereotypic movement disorder and trichotillomania. Prog Neuropsychopharmacol Biol Psychiatry 2002;26:909–12. [7] Hallett M. Physiology of basal ganglia disorders: an overview. Can J Neurol Sci 1993;20:177–83. [8] Kaji R. Basal ganglia as a sensory gating devise for motor control. J Med Invest 2001;48:142–6. [9] Moyanova SG, Rousseva SP. Amphetamine stereotypy in cats and neurotransmitter interactions in the caudate nucleus. II. Effects of intracaudate injection of leu-enkephalin and naloxone. Acta Physiol Pharmacol Bulg 1989;15:47–52. [10] Nambu A, Tokuno H, Takada M. Functional significance of the cortico-subthalamo-pallidal ‘hyperdirect’ pathway. Neurosci Res 2002;43:111–7. [11] Parelkar NK, Wang JQ. Preproenkephalin mRNA expression in rat dorsal striatum induced by selective activation of metabotropic glutamate receptor subtype-5. Synapse 2003;474:255–61. [12] Polgar K, Mate I, Till M, Szekely JI. Modulation by enkephalin analogues and neuroleptics of apomorphine-induced stereotypy and turning behaviour in rats. Neuropharmacology 1987;26: 1309–14. [13] Powell SB, Newman HA, McDonald TA, Bugenhagen P, Lewis MH. Development of spontaneous stereotyped behavior in deer mice: effects of early and late exposure to a more complex environment. Dev Psychobiol 2000;37:100–8. [14] Presti MF, Mikes HM, Lewis MH. Selective blockade of spontaneous motor stereotypy via intrastriatal pharmacological manipulation. Pharmacol Biochem Behav 2003;74:833–9.
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[15] Presti MF, Gibney BC, Lewis MH. Effects of intrastriatal administration of selective dopaminergic ligands on spontaneous stereotypy in mice. Physiol Behav 2004;80:433–9. [17] Salmi P, Sproat BS, Ludwig J, Hale R, Avery N, Kela J, et al. Dopamine D(2) receptor ribozyme inhibits quinpirole-induced stereotypy in rats. Eur J Pharmacol 2000;388:R1–2. [18] Sandman CA, Barron JL, Colman H. An orally administered opiate blocker, naltrexone, attenuates self-injurious behavior. Am J Ment Retard 1990;95:93–102. [19] Scheel-Kruger J, Arnt J, Magelund G, Olianas M, Przewlocka B, Christensen AV. Behavioral functions of GABA in basal ganglia and limbic system. Brain Res Bull 1980;5:261–7. [20] Steiner H, Gerfen CR. Dynorphin regulates D1 dopamine receptormediated responses in the striatum: relative contributions of preand postsynaptic mechanisms in dorsal and ventral striatum demonstrated by altered immediate-early gene induction. J Comp Neurol 1996;376:530–41.
[21] Steiner H, Gerfen CR. Role of dynorphin and enkephalin in the regulation of striatal output pathways and behavior. Exp Brain Res 1998;123:60–76. [22] Steiner H, Gerfen CR. Enkephalin regulates acute D2 dopamine receptor antagonist-induced immediate-early gene expression in striatal neurons. Neuroscience 1999;88:795–810. [23] Werme M, Messer C, Olson L, Gilden L, Thoren P, Nestler EJ, et al. Delta FosB regulates wheel running. J Neurosci 2002;22: 8133–8. [24] You ZB, Herrera-Marschitz M, Nylander I, Goiny M, O’Connor WT, Ungerstedt U, et al. The striatonigral dynorphin pathway of the rat studied with in vivo microdialysis – II. Effects of dopamine D1 and D2 receptor agonists. Neuroscience 1994;63: 427–34. [25] Zanella AJ, Broom DM, Hunter JC, Mendl MT. Brain opioid receptors in relation to stereotypies, inactivity, and housing in sows. Physiol Behav 1996;59:769–75.
Research report
Striatal opioid peptide content in an animal model of spontaneous stereotypic behavior Michael F. Presti∗ , Mark H. Lewis1 Departments of Psychiatry and Neuroscience, University of Florida, 100 S. Newell Dr., P.O. Box 100256, Gainesville, FL 32610, USA Received 27 April 2004; received in revised form 23 July 2004; accepted 5 August 2004 Available online 19 September 2004
Abstract Stereotypic behaviors are associated with a broad spectrum of developmental, psychiatric, and genetic disorders. Several lines of evidence indicate that these abnormal repetitive behaviors may be expressed as a consequence of imbalanced activity along the direct and indirect processing pathways of the basal ganglia. Because the activity of these two pathways is modulated, in part, by the neuropeptides dynorphin and enkephalin, we sought to determine whether spontaneous and persistent stereotypy is associated with alterations in striatal neuropeptide content. Specifically, the present study employed radioimmunoassay to measure dynorphin-A and leu-enkephalin content in the dorsolateral striatum of deer mice exhibiting different levels of spontaneous stereotypic jumping. The results indicate significantly decreased leu-enkephalin content and significantly increased dynorphin/enkephalin content ratios in high-stereotypy mice relative to low-stereotypy mice. Moreover, analyses revealed a significant negative correlation between striatal enkephalin content and frequency of stereotypy as well as a significant positive correlation between the dynorphin/enkephalin content ratio and frequency of stereotypy in these mice. These data are consistent with the hypothesis that spontaneous stereotypic behavior is expressed as a consequence of relative hyperactivity along cortico-basal gangliacortical feedback circuits involving the direct (facilitative) pathway, but suggest that primary perturbations to the indirect (inhibitory) pathway give rise to such imbalanced activity. © 2004 Elsevier B.V. All rights reserved. Keywords: Stereotypy; Dynorphin; Enkephalin; Striatum; Direct pathway; Indirect pathway
1. Introduction Several lines of evidence have implicated basal ganglia nuclei in the mediation of abnormal repetitive behavior. Pharmacological studies have consistently shown that manipulations which enhance activity of the direct (striatonigral) pathway or which inhibit activity of the indirect (striatopallidal) pathway induce motor stereotypy [5,19,3,17]. Conversely, spontaneously emitted stereotypies are attenuated via pharmacological manipulations which inhibit activity of the direct pathway [14] or which enhance activity of the indirect pathway [15]. Moreover, the intense stereotypy induced by ∗
Corresponding author. Tel.: +1 352 294 0427; fax: +1 352 294 0425. E-mail addresses: [email protected] (M.F. Presti), [email protected] (M.H. Lewis). 1 Tel.: +1 352 294 0415; fax: +1 352 294 0425. 0166-4328/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2004.08.003
psychostimulant drugs is associated with increased expression of immediate early genes in striatonigral neurons [2]. Interestingly, transgenic animals that selectively overexpress FosB (an immediate early gene involved in the modulation of neuronal excitability) in striatonigral projection neurons exhibit perseverative wheel running, whereas wheel running is significantly inhibited in animals that overexpress the gene in striatopallidal projection neurons [23]. These studies support the hypothesis that stereotypic and perseverative behaviors are expressed as a result of imbalanced activity along the direct and indirect pathways of the basal ganglia and that this imbalance is characterized by a relative increase in striatonigral tone. Output of the striatonigral and striatopallidal pathways is modulated in part by the neuropeptides dynorphin and enkephalin, respectively, which are specifically localized within the striatal medium spiny neurons comprising these
364
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pathways (for review, see [21]). Furthermore, studies have shown that the expression level of both peptides is positively correlated with activation of their respective basal ganglia pathways [22,24,11]. Given such modulatory interactions, it is reasonable to assume that perturbations to the normal balance of activity along the two main pathways of the basal ganglia will be associated with alterations in neuropeptide content. Specifically, the dynorphin/enkephalin content ratio is expected to correlate positively with increased relative striatonigral tone and negatively with increased relative striatopallidal tone. Numerous studies provide indirect support for an association between the expression of stereotypic behavior and perturbations to endogenous opioid peptide systems. For instance, apomorphine and amphetamine-induced stereotypies are attenuated via intrastriatal administration of both leu- and met-enkephalin [9,12,1]. Furthermore, it has been reported that mu- and kappa-receptor densities are inversely correlated with stereotypy duration in tethered sows [25]. Similarly, human patients exhibiting repetitive self-injurious behavior demonstrate hypersensitivity to opiate administration [6], and such behaviors are attenuated clinically following administration of the mu opioid receptor antagonist naltrexone [18]. Given the above findings, the present study sought to determine if levels of spontaneous stereotypic jumping are correlated with striatal neuropeptide content in the deer mouse model of abnormal repetitive behavior. Specifically, we used radioimmunoassay to measure the concentrations of leuenkephalin and dynorphin-A in the dorsolateral striatum of phenotypically distinct (high stereotypy (HS) and low stereotypy (LS)) mice. Because previous work has shown that this form of stereotypy is dependent on D1 receptor-mediated striatonigral pathway activation [14], we hypothesized a direct relationship between the dynorphin/enkephalin content ratio and the intensity of stereotypy in these animals.
2. Methods 2.1. Animals The study was performed using 34 adult (>2-month-old) male deer mice (P. maniculatus). As previously reported [13], these mice develop high rates of persistent, spontaneously emitted stereotypy which persist at stable levels when housed under standard laboratory conditions. Mice were group caged (three to four same sex mice per cage) in a standard (29 cm × 18 cm × 13 cm) laboratory mouse cage. Rodent chow and water were available ad libitum, and temperature was maintained at 24 ◦ C. Mice were maintained on a 15/9-h light/dark cycle, with lights off at 10:00 h. All procedures were performed in accordance with the guidelines set forth in the NIH Guide for the Care and Use of Laboratory Animals and were approved by the University of Florida Institutional Animal Care and Use Committee.
2.2. Behavioral testing Mice were individually housed in custom plexiglass testing cages (22 cm × 15 cm × 28 cm) and allowed to habituate to the testing environment overnight (20:00–10:00 h). In order to assess behavioral phenotype, mice were continuously monitored with automated activity monitors (Digiscan) for two consecutive 9-h (10:00–19:00 h) active cycles while housed in the custom testing cages. These cages were designed with slotted plexiglass lids to eliminate cagetop activity-related (e.g., feeding) photobeam interruptions. Water was delivered through a small hole on one wall using a custom-fitted spout, and food was placed inside the cage. Both locomotion and vertical stereotypy (jumping or somersaulting) were recorded throughout the testing period and the data were reported in 1-h intervals. Food and water remained available ad libitum throughout testing. After the second day of testing, mice were returned to their home cages and remained there for 1 week prior to being sacrificed for tissue collection. 2.3. Neuropeptide analyses Animals were sacrificed via cervical dislocation 7 days after behavioral testing. Brains were rapidly removed, snapfrozen in isopentane, and stored at −80 ◦ C. An HM 505E cryostat (Microm) was used to remove a 300 m section of the anterior striatum, which was transferred to a freezing stage (FTS Systems) for micropunch dissection of the anterior dorsolateral striatum. This region was selected for analysis because it corresponds with coordinates used for intracerebral drug administration resulting in selective attenuation of spontaneous stereotypy. Thus, the center of the micropunch was targeted to the following stereotaxic coordinates: anterior to bregma 0.5 mm; lateral 2.5 mm; ventral −2.7 mm. Dissected striatal tissue was sonicated in 100 l of Tris extraction buffer consisting of: 20 mM Tris, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 10 M DTT, 1% Triton X-100, and a protease inhibitor cocktail tablet (Roche). Sonicated samples were placed on dry ice and vortexed every 10–15 min for 1 h, and then centrifuged at 13,200 rpm for 15 min at 4 ◦ C. Supernatant was collected and total protein content was determined in duplicate for each sample using a Thermomax microplate reader (Molecular Devices) and bovine serum albumin as the standard. Total protein content was normalized for each sample, and then leu-enkephalin and dynorphin-A concentrations were determined via radioimmunological assay using commercially available kits (Phoenix Peptide) and the protocols provided by the manufacturer. The antibodies supplied with these kits were raised against synthetic peptides and displayed no significant cross-reactivity with other endogenous opioid peptides. 2.4. Data analysis The experimental data were analyzed in two ways. First, groups of low stereotypy and high stereotypy mice (n = 12 and
M.F. Presti, M.H. Lewis / Behavioural Brain Research 157 (2005) 363–368
n = 14, respectively) were generated on the basis of average hourly stereotypic frequency over the 18-h course of behavioral testing. Mice were classified as LS if they exhibited an average of less than 300 jumps per hour, and HS if they exhibited an average of more than 1000 vertical beam interruptions per hour. These criteria were designed to be as stringent as possible while maintaining adequate and approximately equal group sizes. In order to evaluate the hypothesized differences in neuropeptide concentrations between these behaviorally distinct phenotypes, t-tests were performed to compare leu-enkephalin concentrations, dynorphin-A concentrations, and leu-enkephalin/dynorphin-A ratios in the LS and HS groups. Next, Pearson product moment correlation coefficients were calculated in order to determine the extent to which dynorphin, enkephalin, and dynorphin/enkephalin levels correlated with stereotypic intensity and locomotor ac-
365
tivity using all 34 mice. A p-value of 0.05 was adopted as the threshold for assigning statistical significance on all tests.
3. Results 3.1. Relationship between striatal opioid peptides and stereotypy Statistically significant differences were found in the concentrations of striatal neuropeptides associated with LS and HS mice. Specifically, leu-enkephalin content was significantly lower (t = 2.27, d.f. = 18, p = 0.036) and the dynorphinA/leu-enkephalin content ratio was significantly higher (t = 2.781, d.f. = 18, p = 0.012) in HS mice than in LS mice (see Fig. 1, panels a and c). Dynorphin-A concentrations were
Fig. 1. Phenotypic differences in striatal opioid peptide content. (a) LS mice had significantly greater striatal concentrations of leu-enkephalin than HS mice; (b) no significant differences were detected in striatal dynorphin-A concentrations between HS and LS mice; (c) HS mice demonstrated significantly higher dynorphin/enkephalin content ratios than LS mice.
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Fig. 2. Correlation between striatal opioid peptide content and stereotypic frequency. (a) A significant inverse correlation was detected between striatal leuenkephalin concentrations and stereotypic frequency; (b) no relationship was detected between striatal dynorphin-A concentrations and stereotypy level; (c) a significant positive correlation was observed between dynorphin/enkephalin content ratios and stereotypic frequency.
slightly but not significantly (p > 0.05) higher in HS mice as compared to LS mice (see Fig. 1, panel b). The relationship between opioid concentrations and stereotypy was also assessed using all 34 mice. A significant inverse correlation between leu-enkephalin content and stereotypy level was found (r = −0.404, p = 0.036), as well as a significant positive correlation between dynorphin-A/leuenkephalin content ratios and stereotypy level (r = 0.424, p = 0.027). No correlation (r = 0.007, p > 0.05) was detected between dynorphin content and stereotypy level (Fig. 2, panels a through c).
cant (p > 0.05) elevation was found in the enkephalin content of the low locomotion group, and dynorphin/enkephalin content ratios were slightly but not significantly (p > 0.05) higher in the high locomotion group. Similarly, no significant correlations (p > 0.05) were detected between dynorphin concentrations, enkephalin concentrations, or dynorphin/enkephalin content ratios and locomotor activity (r = −0.05, 0.14, and −0.09, respectively).
3.2. Relationship between striatal opioid peptides and locomotion
Data from the present study indicate that the expression of persistent, spontaneously emitted stereotypy in deer mice is associated with alterations of striatal opioid concentrations and, presumably, the balance of activity along basal ganglia output pathways. Specifically, the inverse correlation detected between the intensity of stereotypy (as reflected by frequency) and striatal enkephalin content suggests that stereotypy is exhibited in these mice as a consequence of abnormal hypoactivity of the striatopallidal pathway. Given the sig-
No relationships were detected between dynorphin-A and leu-enkephalin concentrations or dynorphin/enkephalin content ratios and locomotor activity (data not shown). Specifically, no differences were found in dynorphin content between the 13 highest and 13 lowest activity mice with respect to locomotion (p > 0.05), a small but non-statistically signifi-
4. Discussion
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nificant positive correlation between dynorphin/enkephalin content ratios and stereotypy level, these results are also consistent with the interpretation that the chronic expression of stereotypic behavior is associated with relative hyperactivity of the striatonigral pathway. Thus, the data support the hypothesized stereotypy-related imbalance in activity levels of the direct and indirect pathways. Based on current models of general basal ganglia function, the putative role of the direct striatonigral pathway is to facilitate and sustain the expression of cortically initiated motor programs, whereas that of the indirect pathway is to inhibit and terminate, respectively, competing and completed motor programs [10,8,7]. Given these functional roles, it has been suggested that the pathogenesis of both hypokinetic (e.g., Parkinson’s disease) and hyperkinetic (e.g., Huntington’s disease) movement disorders is linked to specific disturbances of these feedback pathways [4]. We hypothesized that stereotypy, like other hyperkinetic disorders, is expressed as a consequence of imbalanced basal ganglia activity characterized by excessive relative striatonigral tone because such an imbalance would serve as a substrate for sustained activation of selected corticospinal motor programs. This hypothesis is supported by the present findings, which suggest that stereotypy becomes increasingly perseverative with increased relative striatonigral tone. These findings are also consistent with reports that wheel-running is significantly more persistent in transgenic mice which selectively overexpress FosB in striatonigral neurons [23]. There is considerable evidence to support the conclusion that the detected perturbations to peptide content reflect alterations to the balance of activity along basal ganglia output pathways. For instance, intrastriatal administration of the selective D1 dopamine receptor agonist SKF-38393, a manipulation that activates the direct pathway, increases local dynorphin release [24] and expression [20]. Similarly, intrastriatal D2-receptor dopamine antagonist administration, a manipulation that activates the indirect pathway, increases enkephalin protein [22] and mRNA expression [11]. These studies demonstrate that intrastriatal dynorphin and enkephalin concentrations are directly related to the activity levels of the striatonigral and striatopallidal pathways, respectively, and suggest that they can be treated as indices of the relative balance of activity along these pathways. Taken together, the present findings support the hypothesis that stereotypic behavior is expressed as a consequence of excessive activity along cortico-basal ganglia-cortical motor feedback circuits involving the striatonigral pathway. Although the differences in neuropeptide content ratios were expected to be driven largely by increased dynorphin expression, the imbalances were primarily attributable to decreased enkephalin expression. Given the putative role of the enkephalin-modulated indirect pathway in terminating direct pathway-sustained motor activity, this finding suggests that behaviors become repetitive and perseverative due to a loss of indirect pathway-mediated inhibitory control. Interestingly, this is consistent with a previous experiment that pointed to
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impaired indirect pathway function in which the deer mice exhibited intense hyperactivity in response to intrastriatal administration of the D2 antagonist raclopride, a drug which reliably induces catalepsy in other rodents [15]. Therefore, we have interpreted the present results to reflect the fact that impaired function of the striatopallidal pathway in deer mice is associated with a relative increase in striatonigral tone and, accordingly, abnormal repetitive and perseverative behavior.
Acknowledgements We are very thankful to Dr. Violetta Zujovic for her expert assistance with protein extraction. This work was also supported in part by an NRSA fellowship to M.F.P. (DA15357).
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