86 Neuroscience Letters, 115 (1990) 86-91 Elsevier Scientific Publishers Ireland Ltd.

NSL 06979

Repeated treatment with amitriptyline or electroconvulsive shock does not affect thyrotropin

releasing hormone receptors in discrete rat brain structures

Edmund Przegalinski, Lucylla Jaworska and Romana Konarska Institute 0[" Pharmacology, Polish Academy o/" Sciences Krako w (Poland)

(Received 6 December 1989: Revised version received 27 February 1990; Accepted 9 March 1990)

Key words: Amitriptyline; Repeated treatment; Thyrotropin-releasing hormone receptor; Thyrotropin- releasing hormone content; Rat brain structure; Electroconvulsive shock

We studied the effect of repeated treatment with amitriptyline (10 mg/kg, p.o., twice daily for 14 days) or electroconvulsive shock (ECS) (once daily for 10 days) on the thyrotropin-releasing hormone (TRH) content and TRH receptors in the cerebral cortex, nucleus accumbens, striatum and septum of the rat. Repeated amitriptyline did not significantly affect the density or affinity of TRH receptors in the examined structures, but caused a marked increase in the TRH content in the striatum and nucleus accumbens. Long-term treatment with ECS reduced the density and affinity of TRH receptors in the septum only, but it increased the TRH concentration in the cerebral cortex and striatum. These results, together with the literature data, indicate that there is no simple relationship between the brain content (and release) of TRH and the functional sensitivity of TRH receptors on one hand, and the density of these receptors

on the other.

A vast body of evidence indicates a link between the antidepressant treatment and thyrotropin-releasing hormone (TRH) [11, 15]. For example, it has recently been reported that repeated treatment with antidepressant drugs or electroconvulsive shock (ECS) alters functional responses to the peptide, which possibly reflects changes in the sensitivity of brain TRH binding sites (receptors). Actually, Bennett et al. [1] reported that the arousal induced by intra-accumbens injection of the TRH analogue CG 3509 to rats was reduced following long-term administration of ami- triptyline, but was increased after repeated treatment with ECS. Sills and Jacobowitz [21] found that prolonged administration of desipramine or nialamide decreased the wet-dog shake response in rats to another TRH analogue, MK 771. Moreover, it has also been reported that repeated treatment with some antidepressant drugs including amitriptyline increases concentration of TRH in some extrahypothalamic brain

Correspondence." E. Przegalinski, Institute of Pharmacology, Polish Academy of Sciences, 12 Smetna

Street, 31 343 Krakow, Poland.

0304-3940/90/$ 03.50 (Q 1990 Elsevier Scientific Publishers Ireland Ltd.

87

structures and in the spinal cord [8, 17]. On the other hand, Lighton et al. [9] found a decrease in the TRH content in the nucleus accumbens and spinal cord after long- term treatment with ECS, though an opposite effect was demonstrated in other brain regions, e.g. in the cerebral cortex and striatum [6, 7]. Importantly, augmentation of the TRH content after amitriptyline was accompanied with an increase in the peptide release [8], whereas a decrease in the TRH level in the nucleus accumbens after ECS occurred concurrently with a reduction in its release [9].

The results of other studies indicate that changes in the endogenous level of TRH may regulate TRH receptors in the central nervous system. In fact, Ogawa et al. [14] and Simasko and Horita [22] reported that chronic administration of the peptide or its analogue reduced the density of TRH receptors in several brain structures, while Sharif et al. [20] and Ogawa et al. [12] demonstrated that the 5,7-dihydroxytrypta- mine-induced depletion of the spinal cord TRH is accompanied with an increase in the numer of TRH receptors in this region.

Therefore it seemed interesting to find out whether repeated treatment with ami- triptyline or ECS, which modifies the functional responsiveness of TRH receptors as well as the brain content and release of the peptide (see above), affects the density and affinity of the TRH receptors in extrahypothalamic brain structures (cerebral cortex, septum, striatum and nucleus accumbens). For comparison, the TRH content in these structures was also assessed.

The experiments were performed on male Wistar rats weighing 220-240 g. The ani- mals were housed at a room temperature (20 - 22°C) on a 12:12 h light-dark cycle (the light on at 07.00 h), with food and water ad libitum.

The rats were treated with amitriptyline hydrochloride (Polfa) in a dose of 10 mg/ kg, p.o., twice daily for 14 days. Control rats were given saline. Other rats were sub- jected to an electroconvulsive shock (ECS) (150 mA, 50 Hz, 0.5 s through ear clip electrodes) or a sham shock once daily for 10 consecutive days. At 24 h after the last treatment with amitriptyline or ECS, the animals were killed by decapitation. Their brains were quickly removed and dissected. Brain structures were immediately frozen on dry ice and stored at -70°C for quantification of the TRH receptor binding and the TRH content.

Pooled tissue samples (nucleus accumbens and septum from 6 rats, striatum and cortex from 3 rats) were homogenized in a sodium phosphate buffer (20 mM, pH 7.4) and centrifugated at 30,000 g for 30 min. The resultant pellets were washed twice by resuspension and centrifugation. The washed membrane pellets were dispersed in a fresh buffer at 50-100 mg of original wet weight/ml and were then immediately as- sayed for specific [3H][3-Me-His2]TRH ([3H]MeTRH) receptor binding [19]. Briefly, the membranes were incubated in 250 pl of the total volume with 0.5-8 nM [3H]MeTRH in the presence or absence of 10 pM of TRH for 5-51/2 h at 0°C. The assay was terminated by a rapid filtration and rinsing with cold 0.9% saline (3 x 3 ml) through a glass fiber filter GF/B (Whatman) using a Millipore filtration mani- fold. The trapped receptor-bound radioactivity was determined by liquid scintillation spectrometry with a counting efficiency of 51%. The specific binding of [3H]MeTRH accounted for 50-65% of the total binding. All the assays were performed in duplicate

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or triplicate. The protein was determined by the method of Lowry et al. [10], using bovine serum albumin as a standard. The amount of the specifically bound [3H]MeTRH was expressed as fmol/mg protein. The resultant data were subjected to the Scatchard analysis. The receptor density (Bmax) and apparent dissociation con- stant (Kd) of [3H]MeTRH were determined after subjecting the data to a linear regres- sion analysis.

Dried ethanol extracts of the brain structures collected from individual rats were used for determination of the TRH content by a radioimmunoassay method (RIA) as previously described [17], using the specific anti-TRH antibodies. The average sensitivity of the RIA was 4 pg/tube. The interassay variation was 11%; the intraassay variation was 2.19%. The results are given as pg/mg protein (mean ± SEM).

The data were evaluated statistically using an analysis of variance (one way) and Dunnet's test.

Repeated treatment with amitriptyline or ECS did not significantly affect the densi- ty or affinity of TRH receptors in the examined structures, with the exception of the septum in which repeated ECS significantly reduced (by ca 30%) the Bmax and Kd val- ues (Table l). Singed ECS did not influence the Bma× values (control: 81.8+2.9, treated: 89.3+_5.9 fmol/mg prot., n=6 ) and Kd values (control: 2.8+_0.4, treated: 2.7 ± 0.3 nM, n = 6) in the latter structure (results not shown). Repeated amitriptyline markedly enhanced the TRH content in the nucleus accumbens and striatum (by 126 and 188%, respectively), whereas repeated ECS produced a similar effect in the cere- bral cortex and striatum (increase by 51 and 181%~ respectively). At the same time repeated ECS reduced the TRH concentration in the nucleus accumbens by ca. 30%, though this effect did not reach the level of statistical significance (Table I).

Our results demonstrate that repeated treatment with amitriptyline or ECS does not significantly affect TRH receptors in a number of extrahypothalamic brain struc- tures (cerebral cortex, septum, nucleus accumbens, striatum), except for a significant reduction in the receptor density in the septum after ECS.

As regards amitriptyline, its lack of effect on TRH receptors in the cerebral cortex and septum is not unexpected, since the drug administered repeatedly neither changes the peptide concentration in these brain regions [8, 17, present study] nor modifies the TRH release in the septal nuclei [8]. On the other hand, the lack of changes in the nucleus accumbens and striatal TRH receptors is somehow surprising for the fol- lowing reasons: (1) repeated administration of amitriptyline induces a dramatic in- crease in the TRH concentration in either structure [8, 17, present study] and in- creases the peptide release in the nucleus accumbens [8]; (2) prolonged treatment with amitriptyline reduces behavioural responses to the injection of a TRH analogue into the nucleus accumbens [1]; (3) long-term administration of TRH or its analogue pro- duces down-regulation and functional subsensitivity of the brain TRH receptors [14, 22]. In the light of the above data down-regulation of the nucleus accumbens and striatal TR H receptors after chronic amitriptyline might be expected. Our negative results in this respect indicate that there is no simple relationship between the brain concentration (and release) of TRH and the functional sensitivity of TRH receptors on one hand, and the density of these receptors on the other.

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A similar dissociation also seems to exist in the case of repeated treatment with ECS. In fact, such treatment was found to reduce the T R H concentration in the nu- cleus accumbens [9, present study) and to increase the peptide level in the cerebral cortex, striatum, amygdala and hippocampus, with no changes in several other re- gions including the septum [6, 7, 9 present study]. Moreover, it was reported that repeated ECS reduces the TRH release in the nucleus accumbens [9] and increases behavioural responses to injection of a T R H analogue into the nucleus accumbens [1]. Thus at least in the nucleus accumbens upregulation of T R H receptors might be expected, the more so as the depletion of T R H in the spinal cord is accompanied with an increase in the number of spinal T R H receptors [12, 20]. However, the pre- sent study has shown that repeated ECS reduces the density of T R H receptors in the septum, but not in the other examined structures including the nucleus accum- bens.

The reason of the above dissociations is unclear, though some literature data seem to support our observations: (1) the seizures induced by ECS or pentylenetetrazol were found to augment the T R H content in some discrete brain structures, without any changes in T R H receptors [13, 18]; (2) an increase in the T R H level in the hippo- campus but not in the striatum, and in the T R H receptor density in the striatum but not in the hippocampus, was shown after amygdaloid kindling [5]; (3) prolonged ad- ministration of T R H or its analogue down-regulates T R H receptors in some brain regions (e.g. in the hypothalamus, hippocampus, amygdala, cerebral cortex), but not in others (striatum, olfactory bulb, thalamus + midbrain, brain stem) [14, 22]; (4) up-regulation of the spinal T R H receptors were found not only after 5,7-dihydroxy- tryptamine, which reduces the T R H level in the spinal cord [12, 20], but also after chronic T R H [16]; however, it is unclear whether the 5,7-dihydroxytryptamine- induced increase in the density of T R H receptors is connected with a reduction in the peptide concentration, since the neurotoxine apart from T R H - also reduces the spinal level of 5-hydroxytryptamine and substance P [2, 12]. All these data, as well as our observations suggest that region-specific differences may exist in TRH receptors, and that in some brain structures these receptors may be regulated differ- ently than in others. Another possibility is the assumed heterogeneity of T R H recep- tors [3], though this hypothesis has recently been questioned [4].

This study was supported by Grant CPBP 06.03.3.4 of the Polish Academy of

Sciences.

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4 Hawkins, E.F., Wade, R. and Engel, W.K., Lack of usefulness of DN-1417 for characterization of a CNS receptor for TRH, J. Neurochem., 49 (1987) 239 243.

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21 Sills, M.A. and Jacobowitz, D.M., Chronic administration of desipramine or nialamide decreases wet- dog shakes in rats produced by the TRH-analog MK 771, Brain Res., 401 (1987) 195-199.

22 Simasko, S.M. and Horita, A., Treatment of rats with the TRH analog MK771. Down-regulation of TRH receptors and behavioral tolerance, Neuropharmacology, 24 (1985) 157-165.