substantia nigra

BRAIN RESEARCH ELSEVIER

Brain Research 644 (1994) 331-334

Short C o m m u n i c a t i o n

Intraventricular insulin increases dopamine transporter mRNA in rat VTA/substantia nigra D i a n n e P. Figlewicz a,,, Patricia Szot b,d Mark Chavez c Stephen C. Woods c Richard C. Veith b,d Departments of Cell Biology and Anatomy and Medicine, Oregon Health Sciences University, 3181 S W Sam Jackson Park Road, Portland, OR 97201, USA J' Departments of Psychiatry and Behavioral Science and " Psychology, Unicersity of Washington, Seattle, WA 98195, USA d Geriatric Research, Education, and Clinical Center, Veterans Administration Medical Center, Seattle, WA 98108, USA (Accepted 25 January 1994)

Abstract

The hormone insulin can down-regulate the function and synthesis of the re-uptake transporter for norepmephrine (NET) in vivo and in vitro. In the present study we tested whether this action of insulin is generalized to another member of the catecholamine transporter family. We determined the level of dopamine transporter (DAT) mRNA expression in the ventral tegmental area (VTA)/substantia nigra compacta (SNc) of rats which were chronically treated with vehicle or insulin via the third cerebral ventricle (i.c.v.). DAT mRNA was significantly elevated in the VTA/SNc of rats treated with insulin, as compared with levels in vehicle-treated rats. This is in contrast to our previous observation that i.c.v, insulin decreases NET mRNA in the rat locus coeruleus, and suggests that insulin may have differential and specific modulatory effects on CNS catecholaminergic pathways.

Key words: Insulin; Ventral tegmentat area (VTA); Substantia nigra compacta; Dopamine transporter; Catecholamine; In situ hybridization

It has been proposed that the hormone insulin acts within the CNS as a neuromodulator which impacts on CNS pathways that regulate energy balance, metabolism, body weight, and food intake [21]. One such pathway is the CNS noradrenergic system. Insulin can decrease the uptake of norepinephrine into adult rat brain synaptosomes and slices [6,17], fetal neuronal cultures [2], and PC12 cells [6]. We have documented that this effect is manifest on both an acute and a chronic basis, and have proposed that one mechanism underlying this effect is the regulation by insulin of the synthesis and synaptic m e m b r a n e concentrations of the norepinephrine transporter (NET; [15]). W h e n the amount of N E T m R N A expression in the locus coeruleus, the major nucleus of noradrenergic cell bodies, was determined in rats treated chronically with i.c.v, insulin or vehicle, a significant decrease of N E T

* Corresponding author. Fax: (1) (503) 494-4253. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSD1 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 0 1 1 4 - R

m R N A expression was observed in insulin-treated animals [7]. This finding is consistent with the functional effects of insulin to decrease N E uptake [2,6,17]. Our basis for proposing such a mechanism was the well-established action of insulin in regulating both the plasma m e m b r a n e recruitment and the synthesis of specific glucose transporter molecules in non-neuronal target cells (e.g. [12]). The effects of insulin on glucose transporters, however, are not generalized to every m e m b e r of the glucose transporter family [3,5]. Therefore. in the present study we tested whether chronic i.c.v, insulin treatment can regulate steady-state m R N A levels for another m e m b e r of the catecholamine transporter family, the dopamine uptake transporter (DAT) [13], in the ventral tegrnental area (VTA) and the substantia nigra compacta (SNc), two regions of rat brain which have dense numbers of dopaminergic cell bodies [4]. Methods for the in vivo treatments were identical to those described previously [11]. Male L o n g - E v a n s rats

D.P. Figlewicz et al. /Brain Research 644 (1994) 331-334

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were implanted with third ventricular cannulas attached to osmotic minipumps that delivered either saline (n = 4; b.wt. = 411 _+ 20 g) or 5 m U / d a y porcine insulin (n = 4; b.wt. = 392 _+ 22 g) for 9 days. As we have previously reported [7], this treatment paradigm results neither in significant peripheral hyperinsulinemia nor hypoglycemia (terminal plasma insulin levels were 127 vs. 133 +_ 42 ~ U / m l ; terminal plasma glucose levels were 158 vs. 213_+ 59 m g / d l for vehicle- vs. insulin-treated animals, respectively). Brains were rapidly removed and frozen. 20 ~ m coronal sections were cut on a cryostat and thaw-mounted onto RNase-free silanized slides (2% 3-aminopropylmethoxysilane in acetone) which were stored at -70°C. A 618 bp portion (nucleotides 1-618) of the DAT m R N A cDNA (generous gift of Dr. Susan Amara, Vollum Institute, Portland, OR) was subcloned back into the Bluescript SKII + / - p h a g e m i d using restriction enzyme B a m H I (Boehringer-Mannheim). The antisense cRNA riboprobe was transcribed from the plasmid linearized with Drall (Boehringer-Mannheim), using T3 polymcrase. Probe was labelled with [3SS]UTP (New England Nuclear, Boston, MA) in a 1 : 2 reaction mixture, purified by extraction using phenol:chloroform and chloroform, and precipitated with 7.5 M ammonium acetate and isopropyl alcohol. The probe was added to a hybridization mix containing 50% formamide, 10% dextran sulfate, 0.3 M NaC1, 10 mM Tris (pH 8.0), 1 mM EDTA, 1 x Denhardt's (0.2% each of bovine serum albumin, Ficoll, and polyvinylpyrrolidone), 0.5 m g / m l yeast tRNA and 200 mM dithiothreitol. The final probe concentration in hybridization solution was 2.0 p m o l / m l . In situ hybridization procedures were performed as previously described by Szot and Dorsa [24]. Briefly, slides were post-fixed in 4% paraformaldehyde and washed in phosphate buffered saline. The slides were then treated with acetic anhydride (0.25% in 0.1 M triethanolamine), dehydrated,

delipidated, and air dried. The 35S-labelled cRNA riboprobe was applied to the tissue, and hybridized overnight at 55°C in a moist chamber. Following incubation, the coverslips were removed and the slides were washed for 30 min in I x SSC (0.15 M NaC1, 0.015 M sodium citrate) at room temperature with shaking. The slides were then washed in RNase buffer (1 M Tris, pH 8.0, 5 M NaC1, 0.5 M EDTA) with 20 ~ g / m l RNase A (Sigma) at 37°C for 30 rain, followed by a 30 min wash in 1 × SSC at room temperature. The slides received three washes at 55°C in 0.1 x SSC for 20 min each, and a final wash in 0.1 × SSC at room temperature for 30 min. Slides were then dehydrated in a graded series of ammonium acetate and alcohol, and allowed to air dry. The slides were dipped in Kodak NTB2 nuclear track emulsion (1 : 1 with 0.6 M ammonium acetate) and stored in the dark at 4°C for 5 days. The slides were developed in Kodak D-19 developer (diluted 1 : 1 with water) at 17°C, rinsed in water, and fixed in Kodak General Fixer. They were stained with Cresyl violet acetate, dehydrated, allowed to air dry, and mounted with coverslips. Specificity in the hybridization signal of the riboprobe was confirmed anatomically by preliminary in situ hybridization assays which demonstrated heavy labelling within the VTA and SNc but no specific labelling within the locus coeruleus. Thus, the DAT cRNA probe does not recognize the mRNA for the NET, in our assay system. From each treatment group, appropriately atlasmatched sections [16] were obtained from four animals. For analysis of the hybridized SSS labelling, the area covered by silver grains under dark-field illumination was quantitated as pixels using a Micro Computer Imaging Device (MCID; Imaging Research Inc., Ont., Canada). Fig. 1 shows dark-field photomicrographs (10 x ) of SsS-labelled riboprobe hybridization to the DAT mRNA of the V T A / S N c of a vehicle-treated rat (left)

, Tj'~ (3

Fig. 1. 10 × magnification dark field image of D A T m R N A hybridization to the V T A / S N c in rats treated with 1VT vehicle (left) or insulin (right).

D.P. Figlewicz et a l. ,/Brain Research (~44 (1994) 3 3 1 - 3 3 4

E F F E C T OF IVT INSULIN ON D A T m R N A IN R A T V T A 8000 6000

h-

4000 2000 0

Control

Insulin *p<0.05

(n=4)

Fig. 2. Quantitation of DAT mRNA from control (n = 4) and insulin-treated (n = 4) VTA/SNc. Data are shown in pixel units.

and an insulin-treated rat (right). i.c.v, insulin infusion resulted in a significant increase of D A T m R N A levels vs. those of the controls. As shown in Fig. 2, pixel number for the integrated V T A / S N c was 8,922 + 687 for vehicle-treated rats, and 15,917 + 2,330 for insulintreated rats ( P < 0.05, unpaired 2-tailed t-test). The results support our previous hypothesis that insulin effects on catecholamine transporters are not generalized but may be specific to the particular catecholamine system [7]. Thus, insulin decreases both the steady-state m R N A levels and activity of the NET, but increases the steady-state m R N A levels and activity of the D A T (we have observed in unpublished studies that acute in vitro incubation of rat striatal slices with physiological concentrations of insulin results in a stimulation of D A uptake). As mentioned above, precedent for the specificity of insulin effects on different members of the transporter family exists within the glucose transporter family [3,5,12]. We have not yet examined insulin effects upon the serotonin or G A B A transporters. The functional implication(s) of this action of insulin upon the D A T remain to be determined. One implication would be that D A would be more rapidly cleared from the synapse when insulin levels are elevated. Currently, no information is available regarding the effects of chronic insulin infusion on extracellular D A concentrations in CNS dopaminergic terminal fields of normal rats. D a t a from diabetic (i.e. insulin-depleted) animals and humans suggest that insulin may be a physiological regulator of CNS dopaminergic pathways, but the specific mechanism(s) underlying dopaminergic dysfunction in diabetes has not been elucidated. In human diabetics, frequency and severity of tardive dyskinesia, a movement disorder associated with abnormal CNS dopaminergic function, has been observed to be increased relative to appropriate control subject populations [9,10]. In experimental diabetes, measurement of D A and its metabolites [25], and a report of

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elevated DA receptors [14], have led to the suggestioH that dopaminergic transmission is decreased with diztbetes. Alternatively, dopaminergic post-synaptic sensitivity and receptor numbers have been reported to be decreased with diabetes [18-20], and in microdialysis studies, elevated basal DA levels in two major dopaminergic projection fields (the nucleus accumbens and the hypothalamus) of diabetic rats have been observed [1,23]. Differences in the severity and duration of diabetes in the various models tested may account for some of the discrepancies in the data. Finally, the ability of the altered metabolic status of diabetic subjects to impact upon CNS catecholaminergic transmission independent of any effects of CNS insulin depletion per se needs to be considered. However, we have obtained preliminary evidence that direct i.c.v, insulin infusion into diabetic rats decreases N E T m R N A levels relative to vehicle-infused diabetic controls [8]. This treatment, similar to the present and previous [7] studies, had no effect on terminal plasma glucose levels, suggesting that there may be a direct CNS role for insulin in the regulation of these transporters. In conclusion, insulin has differential and specific effects upon the steady-state m R N A levels of two CNS catecholamine transporters, the N E T and the DAT. Whether the effects on steady-state m R N A levels are reflected in altered levels of transporter protein within the synaptic membrane, and whether the effects are robust enough to impact upon synaptic concentrations of NE and D A in their terminal fields are critical issues which are under current investigation. The authors gratefully acknowledge the expert technical assistance of Sylvia White, Hong Nguyen, and Ruth Hollingworth. Porcine insulin was the generous gift of Eli Lilly, Indianapolis, IN. This research was supported by N I H Grants D K 40963, DK17844, and the Research Service of the D e p a r t m e n t of Veterans Affairs. [1] Ahmad, Q. and Merali, Z., Diabetic rats display blunted behavioral response to d-amphetamine or dark phase: implications of altered dopamine (DA) function, Soc. Neurosci. Abstr., 18 (1992) 1071. [2] Boyd Jr., F.T., Clarke, D.W. and Raizada, M.K., Insulin inhibits specific norepinephrine uptake in neuronal cultures in rat brain, Brain Res., 398 (1986) 1-5. [3] Burcelin, R., Eddouks, M., Kande, J., Assan, R. and Girard, J., Evidence that GLUT-2 mRNA and protein concentrations are decreased by hyperinsulinemia and increased by hyperglycaemia in liver of diabetic rats, Biochem. J., 288 (1992) 675-679. 14] Cerruti, C., Walther, D.M., Kuhar, M.J. and Uhl, G.R., Dopamine transporter mRNA expression is intense in rat midbrain neurons and modest outside midbrain, MoL Brain Res., 18 (1993) 181-186. [5] Feister, H., Meyer, J., McClain, B., Qulali, M. and Dominguez, J.H., Renal GLUT2 expression in rat models of diabetes, Diabetes, 42 Suppl. 1 (1993) 61A.

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