An investigation of two animal models of anxiety : central administration of corticotropin-releasing factor on the behaviour and neurochemistry of rats, and the effect of pharmacotherapy on spontaneous stereotypical behaviour and NMDA receptor function in mice

Date
2004-12
Journal Title
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Publisher
Stellenbosch : Stellenbosch University
Abstract
ENGLISH ABSTRACT: Corticotropin-releasing factor (CRF) study Background: Hypothalamic pituitary adrenal (HPA)-axis dysfunction is a common symptom of patients with anxiety disorders like posttraumatic stress disorder (PTSO), panic disorder and obsessive-compulsive disorder (OCD). Depressive patients also have HPA-axis dysfunction similar to patients with anxiety disorders. PTSD patients usually show decreased basal plasma cortisol levels whereas OCD and depressive patients show an increase in plasma cortisol. A blunted adrenocorticotropin hormone (ACTH) response to CRF is seen in these patients and mostly an increased level of CRF in their cerebrospinal fluid (CSF). It has been proposed that the CSF level of CRF is a reflection of activity of both hypothalamic and extra-hypothalamic CRF systems. Since the amygdala has been shown to be involved in the endocrine and behavioural response to stress, and CRF is involved in the mediation of this response, we investigated whether chronic elevation of CRF in the amygdala is involved in development of the symptoms of psychiatric disorders. We chronically injected rats with CRF in this area to see what the effect is on their behaviour and HPA-axis response. It has been shown that the hippocampal serotonergic (5-HT) system is involved in both anxiety and depression and that the 5-HT system is regulated by CRF. We therefore measured hippocampal 5- HT1A receptor density and affinity in CRF-injected and control rats. Materials and methods: Male Sprague-Dawley and Wistar rats were stereotaxically implanted with unilateral and bilateral chronic cannulae in the basolateral amygdala (BLA). After recovery unilaterally implanted rats were injected with 10ng (n=7) or 1DOng(n=6) of CRF or saline (n=6) daily for 5 days. Behaviour was tested on day 5 in the elevated plus-maze and open field. Rats with bilateral implants were injected with 100ng CRF (n=19) or saline (n=17) on either side for 5 days. On day 5, behaviour was tested on the elevated plusmaze and open field. Group 1 was tested at baseline levels (n=6 for saline and n=7 for CRF) and group 2 after 5 min. of restraint (n=11 for saline and n=12 for CRF). The stress response of all rats was tested 2 days later. The rats were divided into 3 groups: The first group was decapitated at baseline level (n=6 for each group), the second and third groups restrained for 10 min. and decapitated 15 min. (n=6 for saline and n=7 for CRF) and 60 min. (n=5 for saline and n=6 for CRF) after restraint stress. Blood was collected for plasma ACTH and corticosterone determinations. A group of naïve rats was also included in this experiment to control for the possible effect of the operation (n=6 for each time point). Hippocampi were dissected out and used for 5-HT1A radioligand binding studies. Results: Rats that were unilaterally injected with 1DOngCRF, showed significant increase in the amount of entries into the open arms as well as the amount of time spent in the open arms of the elevated plus-maze compared to controls and rats injected with 10ng CRF. There were no differences between the groups in other parameters of the elevated plus-maze or open field behaviour. There were no significant differences in behaviour of bilateral injected rats compared to controls, but an increase of grooming in the open field was observed in CRFinjected rats that were stressed before behavioural tests. The ACTH and corticosterone response of rats were normal as seen by a significant increase in both concentrations 15 min. after stress and a return to near basal values 60 min. post stress. There were no differences in plasma ACTH concentrations between the groups at any time point. The basal corticosterone level of CRF-injected rats was however significantly lower than controls, but no difference were found 15 or 60 min. post stress. There were no significant differences in hippocampal 5-HT1A receptor densities or affinities of CRF-injected and control rats. Conclusions: We did not observe increased anxiety levels or decreased activity in CRF injected rats. Instead, we observed increased activity in unilateral injected rats. We suspect that the lower dosage of CRF may have this effect on the behaviour of rats, since other authors have also found this result. The lack of differences in the 5-HT1A receptor populations of CRF-injected rats and control rats also confirms this result, since there was no increase in anxiety levels of CRF-injected rats. The chronic elevation of CRF in the SLA caused decreased basal levels of corticosterone in the rats and we speculate that CRF caused adrenal insufficiency although the mechanism is unknown. Stereotypical behaviour study Background: OCD affects 1-2% of the adult human population and is amongst the most common psychiatric disorders. This disorder is characterized by obsessions and compulsions. These compulsions or repetitive behaviours are suggested to be the cause of a hyperactive cortical-striatal-thalamic-cortical (CSTC) circuit in the brain, because of imbalance of the direct and indirect pathways. CSTC pathways are modulated by both 5-HT and dopaminergic (DA) neurons and it has been suggested that a hyperglutamatergic state exists in the frontal cortex of OCD patients. Two types of animal models of stereotypical behaviour are used to investigate neurotransmitter abnormalities related to OCD. These are drug induced stereotypies and environmentally induced (spontaneous) stereotypies. It has been shown that in deermice (Peromyscus manicula tis), drug induced stereotypies are topographically different from spontaneous stereotypies, and our aims were to characterize a deermice model of spontaneous stereotypy for OCD in terms of face, predictive and construct validity. We injected adult deermice, showing spontaneous stereotypies, for a time period of 8 weeks with risperidone (D2/5-HT2 antagonist), citalopram (selective serotonin reuptake inhibitor) and inositol (a metabolic precursor to the phosphatidylinositol second messenger cycle). All these drugs have been shown to improve symptoms of OCD in humans, and we investigated whether the drugs can reduce stereotypies in deermice. Materials and methods: 40 Adult deermice were raised and housed in standard laboratory cages and randomly divided into four groups (6 females and 4 males per group). After baseline recordings of behaviour (3 times per week for a total of 15 min.) the mice were injected daily for 8 weeks with risperidone, citalopram, inositol or saline. Video recordings were made for the 8 week trial and rated afterwards by raters that were blind to the medication status of the animals. A 5 sec. interval scoring system was used for ratings in which the absence or presence of a stereotypy (backward somersault) was noted. After 8 weeks of treatment, the mice were decapitated, brains were dissected and the frontal cortices were stored in liquid nitrogen until radioligand binding studies were performed on NMDA receptors. Results: There were no significant differences in the amount of somersaults between saline injected and drug treated groups when the data was analysed using an ANOVA with repeated measures. There was a significant difference between the control group and drug treated groups at week 8 in male mice when the data was analysed using a Mann-Whitney test. The amount of somersaults shown by saline injected mice increased over the 8 week trial while it stayed more constant in all three drug treated groups. There were no significant differences between the control group and treatment groups in Bmax and Kd values of NMDA receptors in the frontal cortex. There was a trend towards increased receptor densities in all treatment groups compared to the control group and a decrease in affinity in the risperidone group. Conclusions: We found limited evidence for the involvement of both 5-HT and DA systems in the development of spontaneous stereotypical behaviour of deermice. Risperidone, citalopram and inositol were useful in suppressing the increase in somersaults observed in the control group towards week 8 of the trial. This increase was presumably due to stress from handling and injections. The fact that there was a trend towards increased receptor densities in all treatment groups and decreased affinity in the risperidone group also point to the involvement of 5-HT and DA in spontaneous stereotypies. The limitation of this study was small group numbers and excessive stress experienced by the animals.
AFRIKAANSE OPSOMMING: Kortikotropien vrystellingsfaktor (CRF) studie Agtergrond: Hipotalamus-hipofise-adrenale (HPA)-as abnormaliteite is 'n algemene simptoom van pasiente wat gediagnoseer is met verskeie psigiatriese afwykings soos posttraumatiese stres steuring (PTSO), obsessiewe-kompulsiewe steuring (OCD) paniek steuring en depressie. PTSD pasiente toon gewoonlik 'n verlaging in basale kortisol vlakke, terwyl OCD en depressie pasiente "n verhoging in die basale konsentrasie van kortisol in die bloed toon. 'n Verlaagde adrenokortikotropien hormoon (ACTH) respons na toediening van CRF word ook gewoonlik in al die bogenoemde afwykings waargeneem. Pasiente toon ook 'n verhoging in CRF konsentrasies in hul cerebrospinale vloeistof (CSF). Daar is voorheen al bewyse gevind dat die CRF konsentrasie in CSF 'n aanduiding van beide die aktiwiteit van die HPA-as en ander CRF sisteme verteenwoordig. Die amygdala is 'n belangrike deel van die brein wat betrokke is by die endokriene en gedragsrespons op stres. Omdat CRF aangedui is om 'n rol te speel in die stres respons, het ons ondersoek ingestel na die rol van kroniese verhoging van CRF in die amygdala in die ontwikkeling van simptome van psigiatriese steuringe. Rotte is kronies ingespuit in hierdie brein area en die effek daarvan is waargeneem deur na die gedrag en stres respons van die rotte te kyk. Dit is ook al aangedui dat die serotonergiese (5-HT) sisteem in die hippokampus betrokke is in angstigheid en depressie, en dat hierdie sisteem gereguleer word deur CRF. Daarom het ons ook ondersoek ingestelof daar verskille is in die 5-HT1A reseptor populasies van CRF en kontrole rotte. Materiale en metodes: Manlike Sprague-Dawley en Wistar rotte is onderskeidelik met behulp van stereotaksis met chroniese kannules unilateraal en bilateraal geinplanteer in die basolaterale nukleus van die amygdala (BLA). Nadat die rotte herstel het, is rotte met unilaterale inplanterings ingespuit met 10ng (n=7) of 100ng (n=6) CRF of 'n soutoplossing (n=6) daagliks vir 5 agtereenvolgende dae. Hierdie rotte se gedrag is waargeneem op die 5de dag met behulp van die "elevated plus-maze" en "open field". Rotte met bilaterale inplanterings is daagliks ingespuit met 100ng CRF (n=19) of soutoplossing (n=17) vir 5 dae. Die gedrag is op dieselfde manier getoets op die vyfde dag behalwe dat een groep vir 5 min gestres was voor die toets (n=11 vir kontrole en n= 12 vir CRF) terwyl die ander groep rotte waargeneem is by basale vlakke (n=6 vir kontrole en n=7 vir CRF). Die stres respons van hierdie rotte is ook 2 dae later getoets. Die rotte is verdeel in 3 groepe waarvan die eerste groep gedekapiteer is by basale vlakke (n=6 vir kontrole en CRF). Die tweede en derde groepe is blootgestel aan 10 min. stres en gedekapiteer onderskeidelik 15 min. (n=6 vir kontrole en n=7 vir CRF) en 60 min. (n=5 vir kontrole en n=6 vir CRF) na die stres periode. Bloed is opgevang vir plasma ACTH en kortikosteroon bepalings. 'n Groep naiewe rotte is ook ingesluit in hierdie eksperiment om te kontroleer vir die effek wat die operasie kon hê (n=6 vir elke tydstip). Die breine is gedissekteer en hippokampusse verwyder en gestoor in vloeibare stikstof vir radioligand bindingstudies op 5-HT1A reseptore. Resultate: Rotte wat unilaterale CRF inspuitings gekry het, het "n beduidende verhoging getoon in die aantal tyd gespandeer en die aantal kere wat hulle die oop arms van die "elevated plus-maze" binnegegaan het in vergelyking met kontrole rotte. Daar was geen verskil tussen die groepe in enige van die ander parameters waargeneem in die "elevated plus-maze" of "open field" gedrag nie. Daar was ook geen beduidende verskil in enige gedrag van rotte wat bilateraal ingespuit is en kontrole rotte nie, behalwe dat daar "n verhoging was in die totale aantal kere wat die rot versorgingsgedrag getoon het. Die ACTH en kortikosteroon respons van die rotte was normaal in vergelyking met kontrole rotte soos waargeneem deur die beduidende verhoging in konsentrasies 15 minute na stres en die verlaging daarvan na 60 minute. Daar was geen verskille tussen die groepe in die konsentrasies van ACTH nie, maar daar was wel "n beduidend verlaagde basale konsentrasie van kortikosteroon in die CRF groep in vergelyking met kontrole rotte. Daar was ook geen verskil in die 5-HT1A reseptor populasies van die CRF en kontrole rotte nie. Gevolgtrekkings: Ons het geen verhoging in angstigheid of verlaging in aktiwiteitsvlakke waargeneem in die CRF rotte nie. Ons het inteendeel verhoogde aktiwiteitsvlakke waargeneem by CRF rotte wat unilateraal ingespuit was. Ons vermoed dat die lae dosis van CRF hierdie effek teweeg gebring het in die gedrag van die rotte aangesien vorige outeurs ook al hierdie resultaat waargeneem het. Die afwesigheid van verskille in die reseptor populasies van die eRF en kontrole rotte bevestig ons resultaat van die gedragstudie aangesien daar geen verhoging was in anstigheid in eRF rotte nie. Die kroniese verhoging van eRF vlakke in die SLA het In verlaging in basale vlakke van kortikosteroon veroorsaak en, alhoewel die meganisme onbekend is, moontlik die adrenale funksie verlaag. Stereotiepe gedrag studie Agtergrond: 1-2% Van volwassene mense word gediagnoseer met OeD, wat beteken dat dit een van die mees algemene psigiatriese afwykings is. Hierdie afwyking word gekarakteriseer deur obsessies en kompuisies. Dit is bekend dat kompuisies of stereotiepe gedrag voorkom as gevolg van In ooraktiewe korteksstriatum- talamus-korteks (eSTe) sisteem in die brein, as gevolg van In wanbalans tussen die direkte en indirekte paaie. Die eSTe sisteem word gereguleer deur 5-HT en dopamien (DA) neurone en dit is al voorgestel dat In hiperglutamatergiese toestand in die frontale korteks van OeD pasiente bestaan. Twee tipes dier modelle word gebruik om stereotiepe gedrag te bestudeer in terme van neurotransmitter abnormaliteite. Die eerste is dwelm-geinduseerde stereotiepe gedrag en die tweede omgewing-geinduseerde of spontane stereotiepe gedrag. In Vorige studie op muise (Peromyscus maniculatis) het getoon dat dwelm-geinduseerde en spontane stereotiepe gedrag verskillend is in hierdie spesie. Die doel van ons studie was om In muis model vir OCD te karakteriseer. Ons het dus volwasse muise wat spontane stereotiepe gedrag toon vir In tydperk van 8 weke ingespuit met risperidoon, citalopram, inositol of In soutoplossing. Hierdie medikasie word reeds gebruik vir die behandeling van mense met OCD en die doel van ons studie was om te sien of dit ook spontane stereotiepe gedrag in muise kan verminder. Materiale en metodes: 40 Volwasse muise is geteel en aangehou in standaard laboratorium hokke en lukraak verdeel in vier groepe met 6 wyfies en 4 mannetjies per groep. Video opnames is vir 9 weke gemaak vir 5 minute per keer met In totaal van 15 minute per week. Die eerste week is geneem as basislyn en die muise is daagliks ingespuit met die onderskeie medikasie vir die volgende 8 weke. Die onderskeie groepe is ingespuit met risperidoon, citalopram, inositol of In soutoplossing. In 5 Sekonde interval sisteem is gebruik om gedrag waar te neem, en die teenwoordigheid of afwesigheid van stereotiepe gedrag is genoteer. Na 8 weke van behandeling is die muise opgeoffer deur dekapitasie en die breine is gedissekteer en die frontale korteks gebruik vir radioligand studies op NMDA reseptore. Resultate: Daar was geen beduidende verskille in stereotiepe gedrag tussen die kontrole groep en behandelde groepe tussen die basislyn en week 8 wanneer die data geanaliseer is met behulp van In ANOVA met herhaalde waarnemings nie. Daar was wel In beduidende verskil tussen die kontrole groep en die behandelde groepe by mannetjies in week 8 wanneer die data met "n Mann-Whitney toets geanaliseer word. Die aantal stereotiepe gedrag in die kontrole groep het vermeerder oor die 8 week tydperk terwyl dit meer konstant gebly het in behandelde groepe. Daar was geen beduidende verskille in die digtheid en affiniteit van NMDA reseptore van kontrole en behandelde groepe nie. Daar was wel "n neiging tot "n toename in reseptor digtheid by al die behandelde groepe in vergelyking met die kontrole groep en "n afname in affiniteit in die risperidoon groep in vergelyking met die kontrole groep. Gevolgtrekkings: Ons het beperkte bewyse gevind vir die betrokkenheid van beide 5-HT en DA neurotransmitter sisteme in die ontwikkeling van spontane stereotiepe gedrag in die muise. Risperidoon, citalopram en inositol het die toename in stereotiepe gedrag, soos waargeneem in die kontrole groep, onderdruk. Hierdie toename in stereotiepe gedrag was waarsynlik as gevolg van uitermatige stres wat die muise ervaar het as gevolg van hantering en inspuitings. Die feit dat daar "n neiging tot toename in reseptor digthede in al die behandelde groepe en afname in affiniteit was in die risperidoon groep dui ook op die betrokkenheid van 5-HT en DA in spontane stereotiepe gedrag. Die beperkings van hierdie studie was die klein groep nommers en uitermatige stres wat die muise ervaar het.
Description
Thesis (MScMedSc)--Stellenbosch University, 2004.
Keywords
Anxiety, Corticotropin releasing hormone, ACTH, Obsessive-compulsive disorder, Methyl aspartate, Dissertations -- Medicine, Theses -- Medicine
Citation