In situ hybridization histochemistry as a method to assess GABAAreceptor subunit mRNA expression following chronic alprazolam administration

Abstract

Previous work in our laboratory has demonstrated region-specic effects for chronic alprazolam on binding and function at the GABA_A receptor. The present study evaluated regional changes in mRNA expression of several subunits of the GABA_A receptor following chronic alprazolam administration that might underlie these effects. Mice received alprazolam (2 mg/kg/day) or vehicle via subcutaneously implanted osmotic pumps for 1, 7, 14 or 28 days. In situ hybridization histochemistry was performed on tissue sections using [³⁵S]dATP oligonucleotide probes corresponding to the a₁ and g₂ subunits of the GABA_A receptor. Specic hybridization was clearly demonstrated and a₁ subunit mRNA expression in frontoparietal cortex (layers IIIV) on day 1 of infusion was reduced in animals receiving alprazolam compared to vehicle. On subsequent days, there were no alterations in the levels of a₁ subunit mRNA in the frontoparietal cortex, hippocampus or dentate gyrus. Expression of g₂subunit mRNA was increased on day 1 in the frontoparietal cortex (layer VI), hippocampus and dentate gyrus. mRNA expression was also increased in the dentate gyrus on day 28 of infusion. Comparison of the present study with the results of chronic treatment with other benzodiazepines clearly demonstrates that the pattern of mRNA subunit alterations obtained is both treatment- and region-specic. This makes a de¢nitive conclusion regarding benzodiazepines and their interactions with GABA_A receptors di¤cult at best.

Keywords

alprazolam benzodiazepine GABA in situ hybridization histochemistry mRNA

Get full access to this article

View all access options for this article.

References

Ausubel F M , Brent R , Kingston R E , Moore D D , Seidman J G , Smith JA , Struhl K (1989) Current protocols in molecular biology.Wiley & Sons, New York

Ballenger JC , Burrows G D , DuPont R L , Lesser I M , Noyes R , Pecknold JC , Rifkin A , Swinson R P (1988) Alprazolam in panic disorder and agoraphobia: results from a multicenter trial . Arch Gen Psychiatry 45: 413–422

Ciraulo DA , Barnhill JA , Boxenbaum HG , Greenblatt D J , Smith R B (1986) Pharmacokinetics and clinical effects of alprazolam following single and multiple oral doses in patients with panic disorder . J Clin Pharmacol 26: 292–298

Feighner J P , Aden G C , Fabre L F , Rickels K , Smith W T (1983) Comparison of alprazolam, imipramine and placebo in the treatment of depression . J Am Med Assoc 249: 3057–3064

Fyer A J , Liebowitz M R , Gorman J M , Campeas R , Levin A , Davies S O , Goetz D , Klein DF (1987) Discontinuation of alprazolam treatment in panic patients . Am J Psychiatry 144: 303–308

Gri¤ths R R , Sannerud CA , Ator NA , Brady J V (1992) Zolpidem behavioral pharmacology in baboons: self-injection, discrimination, tolerance and withdrawal . J Pharmacol Exp Ther 260: 1199–1208

Henniger C , Saito N , Tallman J F , Garrett M P , Vitek K M , Duman R S , Gallager DW (1990) Effects of continuous diazepam administration on GABAA subunit mRNA in rat brain . J Mol Neurosci 2: 101–107

Impagnatiello F , Pesold C , Longone P , Caruncho H , Fritschy J M , Costa E , Guidotti A (1996) Modications of g-aminobutyric acidAreceptor subunit expression in rat neocortex during tolerance to diazepam . Mol Pharmacol 49: 822–831

Kang I , Miller L G (1991) Decreased GABAA receptor subunit mRNA concentrations following chronic lorazepam administration . Br J Pharmacol 103: 1285–1287

10.

Lopez F , Miller L G , Greenblatt D J , Paul S M , Shader R I (1988) Low-dose alprazolam augments motor activity in mice . Pharmacol Biochem Behav 30: 511–513

11.

Lopez F , Miller L G , Greenblatt D J , Chesley S , Schatzki A , Shader R I (1990) Chronic administration of benzodiazepines ö V. Rapid onset of behavioral and neurochemical alterations after discontinuation of alprazolam . Neuropharmacology 29: 237–241

12.

Macdonald R L , Olsen RW (1994) GABAA receptor channels . Ann Rev Neurosci 17: 569–602

13.

Miller JA (1991) The calibration of 35S or 32P with 14C-labeled brain paste or 14C-plastic standards for quantitative autoradiography using LKB Ultro¢lm or Amersham Hyper¢lm . Neurosci Lett 121: 211–214

14.

Miller L G , Greenblatt D J , Barnhill J G , Deutsch S I , Shader R I , Paul S M (1987) Benzodiazepine receptor binding of triazolobenzodiazepines in vivo: increased receptor number with low-dose alprazolam . J Neurochem 49: 1595–1601

15.

Miller L G , Woolverton S , Greenblatt D J , Lopez F , Roy R B , Shader R I (1989) Chronic benzodiazepine administration. IV. Rapid development of tolerance and receptor downregulation associated with alprazolam administration . Biochem Pharmacol 38: 3773–3777

16.

O'Donovan MC , Buckland P R , Spurlock G , McGu¤n P (1992) Bidirectional changes in the levels of messenger RNAs encoding g-aminobutyric acidA receptor a subunits after £urazepam treatment . Eur J Pharmacol 226: 335–341

17.

Olsen RW , Tobin A J (1990) Molecular biology of GABAA receptors . FASEB J 4: 1469–1480

18.

Pesold C , Caruncho H J , Inpagnatiello F , Berg M J , Fritschy J M , Guidotti A , Costa E (1997) Tolerance to diazepam and changes in GABAA receptor subunit expression in rat neocortical areas . Neuroscience 79: 477–487

19.

Pritchett DB , Luddens H , Seeburg P H (1989a) Type I and type II GABAA-benzodiazepine receptors produced in transfected cells . Science 245: 1389–1392

20.

Pritchett DB , Sontheimer B , Shivers S , Ymer S , Kettenmann P , Scho¢eld R , Seeburg P H (1989b) Importance of a novel GABAAreceptor subunit for benzodiazepine pharmacology . Nature 338: 582–585

21.

Ramsey-Williams VA , Carter DB (1996) Chronic triazolam and its withdrawal alters GABAA receptor subunit mRNA levels: an in situ hybridization study . Mol Brain Res 43: 132–140

22.

Shader R I , Greenblatt D G (1993) Use of benzodiazepines in anxiety disorders . N Engl J Med 328: 1398–1405

23.

Sieghart W (1995) Structure and pharmacology of g-aminobutyric acid A receptor subtypes . Pharmacol Rev 47: 181–234

24.

Tietz E I , Huang X , Weng X , Rosenberg HC , Chiu T H (1994) Expression of a1, a5 and g2 GABAA receptor subunit mRNA measured in situ in rat hippocampus and cortex following chronic £urazepam administration . J Mol Neurosci 4: 277–292

25.

Young I W S (1989) In situ hybridization histochemical detection of neuropeptide mRNAs using DNA and RNA probes. In Conn PM (ed.), Methods in enzymology: neuroendocrine peptides 3. Academic Press, Orlando, FL

26.

Zhao TJ , Chiu T H , Rosenberg HC (1994) Reduced expression of g-aminobutyric acid type A/benzodiazepine receptor g2 and a5 subunit mRNAs in brain regions of £urazepam-treated rats . Mol Pharmacol 45: 657–663