Increase in calcitonin mRNA levels in rats at high risk of C cell tumours is genetically determined

C-cells tumours are frequent (50%) in old WAG/Rij rats. In comparison to the original Wistar strain, three month old WAG/Rij rats are characterized by higher calcitonin synthesis and secretion, in addition to a genetically transmitted loss of calcitonin binding sites in the outer renal medulla. In order to determine if the increase of calcitonin gene expression is also of genetic origin, we quantified calcitonin and its specific messenger in the thyroid glands of second generation (Wistar x WAG/Rij) hybrids. These parameters ranged from low Wistar like to higher WAG/Rij like values. The amount of calcitonin messenger in the thyroid was highly correlated to the release of the hormone in plasma elicited by a calcium challenge and inversely correlated with the number of its binding sites in the kidney. Our results suggest that an enhanced expression of the calcitonin gene is genetically transmitted, probably as a consequence of the mutation involved in the loss of renal calcitonin binding sites. It may represent the first event leading to malignancy.     

Loss of calcitonin binding in rat is not related to calcitonin receptor gene abnormality.

C-cell tumors occur frequently (50%) in old WAG/Rij rats. Interestingly, genetically transmitted loss of CT binding sites in the kidney has also been demonstrated in WAG/Rij rats. To determine if these issues are resulted from mutation of calcitonin receptor (CTR), we analyzed the CTR genomic abnormality in WAG/Rij rat. We demonstrated that both Wistar and WAG/Rij rats expressed type-C1a CTR by RT-PCR analysis and their mRNA expressions were approximately equal by Northern blotting analysis. Direct sequence of RT-PCR products for CTR showed no different nucleotide sequences between the two strains. There were three polymorphisms at the first transmembrane domain and the fourth intracellular membranes, which are different from Sprague-Dawley rat. We concluded that the loss of CT binding in WAG/Rij rat is not related to CTR gene abnormality. Abnormal system of CTR amino acid modification may be occurred in WAG/Rij rat.     

Low dose of disulfiram and l-dopa evoked synergistic modification in behavior of Wistar and WAG/Rij rats

Comparative analysis of behaviors of two rat strains, Wistar and WAG/Rij, was performed. No behavioral differences between Wistar and WAG/Rij were found in the emotional resonance test. Disulfiram injection produced similar effects in both rat strains. Animals of the first group (with slow acquisition of emotional resonance reaction) transformed into the animals of the second group (with fast acquisition). Passive avoidance conditioning was successfully reproduced in Wistar and was significantly impaired in WAG/Rij. A low dose of disulfiram injected before or immediately after conditioning substantially improved the reproduction to a greater extent in WAG/Rij than Wistar strains thus eliminating in interstrain differences. Active avoidance conditioning was more successful in WAG/Rij than in Wistar rats However, on the next day conditioning in WAG/Rij was substantially impaired. Administration of the low dose of disulfiram or L-DOPA prior to conditioning impaired the acquisition but improved the reproduction on the following day in both strains, but disulfiram injection after conditioning improved conditioning in WAG/Rij to a greater extent than in Wistar. Thus, the pharmacologic enhancement of the reward system substantially changed animal behavior and improved memory consolidation.     

Stress, glucocorticoids and absences in a genetic epilepsy model

Although stress can alter the susceptibility of patients and animal models to convulsive epilepsy, little is known about the role of stress and glucocorticoid hormones in absence epilepsy. We measured the basal and acute stress-induced (foot-shocks: FS) concentrations of corticosterone in WAG/Rij rats, non-epileptic inbred ACI rats and outbred Wistar rats. The WAG/Rij strain is a genetic model for absence epilepsy and comorbidity for depression, which originates from the population of Wistar rats and, therefore, shares their genetic background. In a separate experiment, WAG/Rij rats were exposed to FS on three consecutive days. Electroencephalograms (EEGs) were recorded before and after FS, and the number of absence seizures (spike-wave-discharges, SWDs) was quantified. Both WAG/Rij rats and ACI rats exhibited elevated basal levels of corticosterone and a rapid corticosterone increase in response to acute stress. The WAG/Rij rats also displayed the most rapid normalization of corticosterone during the recovery phase compared to that of ACI and Wistar rats. FS had a biphasic effect on SWDs; an initial suppression was followed by an aggravation of the SWDs. By the third day, this aggravation of seizures was present in the hour preceding FS. This increase in SWDs may arise from anticipatory stress about the upcoming FS. Together, these results suggest that the distinct secretion profile of corticosterone found in WAG/Rij rats may contribute to the severity of the epileptic phenotype. Although the acute stressor results in an initial suppression of SWDs followed by an increase in SWDs, stress prior to a predictable negative event aggravates absences.     

Anxiety and behavior in WAG/Rij strain rats with genetically induced absence attacks

Behavior of nonlinear rats and animals from Wistar and WAG/Rij (with inborn generalized absence epilepsy) strains was examined in the elevated plus-maze and the hole board. WAG/Rij rats demonstrated low exploratory behavior in both tests. In the elevated plus-maze, WAG/Rij rats were more balanced and more anxious than Wistar and nonlinear rats. Administration of ethosuximide completely eliminated spike-wave discharges but did not change behavioral interstrain differences. Since the spike-wave patterns develop in WAG/Rij at the age of 3 months, the behavior of young (2-moth-old) pups from different strains was compared and significant differences were revealed. Correlation between the genetically defined features (spike-wave discharges) and behavioral peculiarities in WAG/Rij rats is supposed.     

Are WAG/Rij rats with genetic absence epilepsy anxious?

Behavior of susceptible and non-susceptible to audiogenic (convulsive) seizures rats from inbred WAG/Rij strain, genetically predisposed to absence epilepsy, and outbred Wistar strain, genetically not predisposed to absence epilepsy, was compared to assess the level of anxiety (in open field, light-dark choice and elevated plus-maze tests) and the level of depressiveness (in the sucrose consumption and forced swimming tests). Increased level of anxiety was found only in susceptible to audiogenic seizures rats both from WAG/Rij and Wistar strain, but increased level of depressiveness was found only in WAG/Rij strain rats as compared with Wistar rats independently of their susceptibility to audiogenic seizures. Results suggest that increased depressiveness in WAG/Rij strain rats is associated with absence epilepsy but increased anxiety with susceptibility to audiogenic seizures.     

Behavioral characteristics of female Wag/Rij rats

Behavior of male and female WAG/Rij and Wistar rats was compared in the tests assessing the level of anxiety (light-dark choice, open field) and depression-like state (sucrose intake and preference, forced swimming). Females of WAG/Rij rats like males of the same strain exhibited symptoms of depression-like behavior: increased immobility in the forced swimming test and decreased sucrose intake and preference (anhedonia). In contrast to males, females of WAG/Rij rats displayed more distinct signs of increased anxiety as compared to Wistar rats. Both WAG/Rij and Wistar females exhibited increased locomotor and exploratory activity in the open field as compared to males.     

Dopamine-dependent character of depressive-like behavior in WAG/Rij rats with genetic absence epilepsy

Placebo-treated WAG/Rij rats (as compared to normal Wistar rats without seizure pathology) exhibited depressive-like behavior similar to that of intact rats of the same strain: decreased exploratory activity in the open field test, increased immobility in the forced swimming test, decreased sucrose consumption and preference (anhedonia). Chronic injection of tricyclic antidepressant imipramine (15 mg/kg. i.p., for 15 days) exerted a therapeutic (antidepressant) effect on depressive-like behavior in WAG/Rij rats. After cessation of antidepressant therapy, the behavior of WAG/Rij rats didn't significantly differ from that of Wistar rats. Acute (single) injection of selective D2/D3 dopamine receptor antagonist raclopride (100 microg/kg, i.p., 15 min prior to behavioral testing) aggravated the symptoms of depressive-like behavior and suppressed antidepressant effect of chronic injection of imipramine in WAG/Rij rats, whereas it didn't exert a substantial effect on behavior of Wistar rats. Injection of D2/D3 dopamine receptor agonist Parlodel (bromocriptine) counteracted the depressive-like behavior in WAG/Rij rats and didn't exert substantial influence on behavior of Wistar rats with the exception of a decrease in immobility time in the forced swimming test. Injections of imipramine and raclopride didn't exert significant influences on the level of general locomotor activity and anxiety both in WAG/Rij and Wistar rats. The results demonstrate the dopamine-dependent character of depressive-like behavior in WAG/Rij rats, and indicate possible involvement of dopamine D2-like receptors in mediation of the antidepressant effect of imipramine on genetically determined depressive-like behavior in WAG/Rij rats.     

Strain differences in kidney copper concentrations of male rats

The copper concentrations of the kidneys of male rats of six inbred (BN, F344, LEW, SHR, WAG/Cpb, and WAG/Rij) and one random-bred Wistar strain were determined. In inbred rats the mean concentration varied between strains and ranged from 7.10 μg/g for F 344 to 23.48 μg/g for WAG/Cpb. The calculated coefficient of genetic determination (g²) was 0.88. A remarkable discrepancy was found between the two WAG inbred strains; the WAG/Cpb had a 2.5 times higher kidney Cu concentration than the WAG/Rij. Kidney Cu concentrations of random-bred rats varied considerably; the coefficient of variation of the means was 28 and 34% in two samples taken with a 1-yr interval, respectively, indicating inhomogeneity within the population. The results indicate that the individual differences in kidney Cu concentration have a genetic basis.     

Depression-like changes in behavior and c-fos gene expression in dopaminergic brain structures in WAG/Rij rats

In WAG/Rij rats with genetic absence epilepsy, inborn changes in behavior were observed such as decreased level of locomotion, exploratory activity, and grooming reactions in the open-field test, increased immobility in the forced-swimming test, and decreased sucrose consumption (anhedonia) as compared to Wistar rats completely lacking in seizure pathology. These behavioral alterations in WAG/Rij rats resemble the symptoms of human depression (psychomotor retardation, depressed mood, and anhedonia). No significant behavioral changes were found in the light-dark choice, social interaction, and elevated plus-maze tests. This suggests the absence of increased anxiety in WAG/Rij rats. In contrast to Wistar, WAG/Rij rats were sensitive only to chronic treatment with antidepressant imipramine like depressive patients. Behavioral "despair" induced by forced swimming led to C-fos gene expression in three brain structures (frontal cortex, nucleus accumbens, and striatum), which are, respectively, terminal regions of three dopaminergic brain systems (mesocortical, mesolimbc, and nigrostriatal). c-fos gene expression in the brain of WAG/Rij rats was substantially different from that in the brain of Wistar rats in both intensity (in WAG/Rij the c-fos gene expression was higher than in Wistar rats in all involved brain structures) and its distribution between the structures. The results suggest that WAG/Rij strain is a new experimental (genetic) model of absence epilepsy-related depression unassociated with increased anxiety.     

Maternal care exerts disease‐modifying effects on genetic absence epilepsy and comorbid depression

WAG/Rij rats, a genetic animal model of absence epilepsy with comorbidity of depression, exhibit behavioral depression‐like symptoms and spontaneous generalized spike‐wave discharges (SWDs) in the EEG at the age of 6 to 8 months. The aim of the present study was to test the hypothesis that maternal care is an environmental factor which, along with genetic predisposition, may contribute to the expression of absence seizures and depression‐like comorbidity later in life. To achieve this, a cross‐fostering procedure was used. EEG and behavior in the forced swimming test were analyzed in WAG/Rij and Wistar offspring reared by their own mothers (non‐cross‐fostered), foster mothers of the same strain (in‐fostered) or another strain (cross‐fostered) at the age of 7 to 8 months. Maternal care and forced swimming test behavior were assessed in the dams. WAG/Rij mothers showed depression‐like behavior and reduced maternal care irrespective of litter size and litter composition (own or foster pups) compared with Wistar dams. WAG/Rij offspring reared by Wistar dams with a high level of maternal care exhibited less and shorter SWDs and reduced depression‐like comorbidity in adulthood compared with age‐matched WAG/Rij offspring reared by their own or foster WAG/Rij mothers with a low level of maternal care. Moreover, rearing by Wistar mothers delayed the onset of absence epilepsy in WAG/Rij rats. Adoption by WAG/Rij dams did not change EEG and behavior in Wistar rats. Our study demonstrates that improvement of early care‐giving environment can be used as a disease‐modifying treatment to counteract epileptogenesis and behavioral comorbidities in genetic absence epilepsy.     

Possible mechanisms for forming typological features of behavior of WAG/Rij line rats

Typological behavior reactions of WAG/Rij rats were studied from the standpoint of divergent modulatory integration hypothesis. This rat strain has a genetically determined dominant dysfunction of the benzodiazepine system of the thalamic nuclei. This disorder provokes an epileptiform disease such as absence epilepsy. It was suggested that the dysfunction of this system would result in a modification of the modulatory systems, which support the motivation states of escape and avoidance reactions as well as of the modulatory systems, which form the emotional states. Modifications of these states are the background of typological behavioral features of WAG/Rij rats. It was shown that WAG/Rij have the lower threshold of the development of haloperidol catalepsy, higher levels of fear and depression. On the first day of training in a shuttle box, WAG/Rij rats demonstrated better avoidance performance than Wistar rats. On the second and 28th days, the amnestic effect of the epileptiform disease was observed. The amnestic effect was also observed after passive avoidance conditioning. The results are discussed in terms of the modulatory integratin hypothesis.     

Expression of the first calcitonin/CGRP gene in spontaneous and transplanted rat medullary thyroid carcinoma: a comparison of dot-blot analysis, in situ hybridization, and immunohistochemistry.

Calcitonin (CT) and calcitonin gene-related peptide (CGRP) are encoded by a single gene, the CALC-I gene. They are expressed in the thyroid and in the nervous system by alternative splicing of the pre-messenger RNA derived from the CALC-I gene. In medullary thyroid carcinoma (MTC), a malignancy derived from the calcitonin-producing C-cells in the thyroid, production of calcitonin and CGRP is a common feature. We investigated the CT and CGRP production of four spontaneous MTCs transplanted three to four times and 14 MTC lines transplanted for several years in WAG/Rij rats, a strain with hereditary MTC. The expression of CT and CGRP in the spontaneous and in the transplanted tumors was studied by means of RNA in situ hybridization (RISH), dot-blot analysis, and immunohistochemistry. A down-regulation of CT production in transplanted compared with spontaneous tumors was observed, but an inverse relation between CT and CGRP mRNA content in both spontaneous and transplanted tumors was not observed. In this study, RISH proved to be as sensitive as dot-blot analysis to detect gene expression in tissue samples. The different approaches of analyzing the gene expression in tissue samples (the cellular localization of gene expression by ISH vs the analysis of an extract of a total tissue sample with dot-blot analysis) showed that each technique is equal in value and that they are complementary to each other.     

Neuroactive steroids and GABAA receptor plasticity in the brain of the WAG/Rij rat, a model of absence epilepsy

The role of neuroactive steroids and GABA(A) receptors in the generation of spontaneous spike-and-wave discharges (SWDs) was investigated in the WAG/Rij rat model of absence epilepsy. The plasma, cerebrocortical, and thalamic concentrations of the progesterone metabolite 3alpha-hydroxy-5alpha-pregnan-20-one (3alpha,5alpha-TH PROG) were increased in the WAG/Rij rat at 2 months of age compared with those in control (Wistar) rats. In contrast, the brain and peripheral levels of 3alpha,5alpha-tetrahydrodeoxycorticosterone (3alpha,5alpha-TH DOC) did not differ between the two rat strains at this age. At 6 months of age, when absence epilepsy worsens in WAG/Rij rats, the plasma concentration of 3alpha,5alpha-TH PROG remained high whereas that of 3alpha,5alpha-TH DOC had increased, the cerebrocortical levels of both 3alpha,5alpha-TH PROG and 3alpha,5alpha-TH DOC had increased, and the thalamic concentrations of these metabolites had decreased. At 6 months of age the expression of the alpha(4) and delta subunits of the GABA(A) receptor in relay nuclei was increased. Finally, chronic stress induced by social isolation elicited a reduction in the amount of 3alpha,5alpha-TH PROG in the thalamus of 2-month-old WAG/Rij rats that was associated with a reduction in the number and overall duration of SWDs at 6 months of age. Absence epilepsy in the WAG/Rij rat is thus associated with changes in the abundance of neuroactive steroids and in the expression of specific GABA(A) receptor subunits in the thalamus, a brain area key to the pathophysiology of this condition.     

The Modulatory Effect of Metabotropic Glutamate Receptor Type-1α on Spike-Wave Discharges in WAG/Rij Rats

Modulatory function of metabotropic glutamate type 1 (mGlu1) receptors plays a crucial role in the pathophysiology of some neurological disorders, including schizophrenia and epilepsy. In this study, the expression of mGlu1α receptors in the thalamic nuclei was assessed during development of absence seizures in the WAG/Rij rats, a valid genetic animal model of absence epilepsy. In addition, the effect of pharmacological modulation of mGlu1α receptors in the laterodorsal (LD) nucleus of the thalamus on the characteristic features of bioelectrical brain activities in the WAG/Rij rats was assessed. The expression of mGlu1α receptors in the LD was assessed in four experimental groups of both WAG/Rij and Wistar rats with 2 and 6 months of age. Agonist and antagonist of mGlu1α receptors were infused in LD in the six months old WAG/Rij (epileptic) rats. The protein level of mGlu1α receptors in the thalamus of the 6-month-old WAG/Rij rats was lower than non-epileptic animals. In addition, the distribution of mGlu1α receptors in different thalamic nuclei was lower in the 6-month-old WAG/Rij compared to age-matched Wistar rats. The gene expression of mGlu1α receptor was also significantly lower in 6-month-old WAG/Rij rats in the LD compared to other animal groups. The microinjection of mGlu1α receptors agonist and antagonist in the LD reduced the duration of spike-wave discharges (SWDs) and increased the amplitude and duration of SWDs, respectively, in 6-month-old WAG/Rij rats. The alterations of mGlu1α receptors expression in the thalamus of epileptic WAG/Rij rats as well as its modulatory effects in the generation of SWDs suggest the potential of mGlu1 receptors as a therapeutic target in absence epilepsy.     

Synaptic hyperexcitability of deep layer neocortical cells in a genetic model of absence seizures

We used sharp-electrode, intracellular recordings in an in vitro brain slice preparation to study the excitability of neocortical neurons located in the deep layers (>900 microm from the pia) of epileptic (180-210-days old) Wistar Albino Glaxo/Rijswijk (WAG/Rij) and age-matched, non-epileptic control (NEC) rats. Wistar Albino Glaxo/Rijswijk rats represent a genetic model of absence seizures associated with generalized spike and wave (SW) discharges in vivo. When filled with neurobiotin, these neurons had a typical pyramidal shape with extensive apical and basal dendritic trees; moreover, WAG/Rij and NEC cells had similar fundamental electrophysiological and repetitive firing properties. Sequences of excitatory postsynaptic potentials (EPSPs) and hyperpolarizing inhibitory postsynaptic potentials (IPSPs) were induced in both the strains by electrical stimuli delivered to the underlying white matter or within the neocortex; however, in 24 of 55 regularly firing WAG/Rij cells but only in 2 of 25 NEC neurons, we identified a late EPSP that (1) led to action potential discharge and (2) was abolished by the N-methyl-D-aspartate (NMDA) receptor antagonist 3,3-(2-carboxypiperazine-4-yl)-propyl-1-phosphonate (20 microM; n = 8/8 WAG/Rij cells). Finally, we found that the fast and slow components of the stimulus-induced IPSPs recorded during the application of glutamatergic receptor antagonists had similar reversal potentials in the two strains, while the peak conductance of the fast IPSP was significantly reduced in WAG/Rij cells. These findings document an increase in synaptic excitability that is mediated by NMDA receptors, in epileptic WAG/Rij rat neurons located in neocortical deep layers. We propose that this mechanism may be instrumental for initiating and maintaining generalized SW discharges in vivo.     

The Ret proto-oncogene in the WAG/Rij rat strain: an animal model for inherited C-cell carcinoma?

WAG/Rij rat strain has been suggested as an animal model for the study of inherited human medullary thyroid carcinoma (MTC), due to its high incidence of spontaneous C-cell thyroid tumours. Although the role of the Ret proto-oncogene mutations, as responsible for human MTC, is well established, nothing has been published concerning this putative animal model. Based upon the previously reported rat Ret sequence, exons 10, 11, 13, 14, 15, and 16, known to carry activating mutations in humans, have been analysed in the WAG/Rij rat by PCR, single strand conformational polymorphism (SSCP) and direct sequencing. Neither the germline nor MTC samples showed any Ret sequence difference in the exons when analysed in comparison to a non-MTC-susceptible rat strain. Our results indicate that Ret exons relevant in humans are not involved in WAG/Rij rat MTC, as expected, and this questions the validity of this strain as a model for the human disease, and suggests there must be additional mechanisms for the genesis and progression of rat MTC.     

The influence of neonatal ketamine injection on pain sensitivity and audiogenic seizures in adult rats

The remote effects of neonatal (on the 3d-to-9th postnatal days) ketamine injections (10 and 50 mg/kg in 20 microliters of distilled water, s.c.) were analyzed in adult Wistar, WAG/Rij, and KM (a strain with high audiogenic sensitivity) rats. Both ketamine and water injections increased pain sensitivity in adult rats. Neonatally injected water increased the mean score of seizures in Wistar and WAG/Rij, whereas ketamine water solution injected in the dose of 50 mg/kg did not change the seizure intensity (as compared to the intact control). Consequently, ketamine significantly reduced the mean score of the audiogenic seizure fit without change in its latency. In highly sensitive KM rats the neonatally injected ketamine (50 mg/kg) significantly shortened the mean latency of the fit onset, and fit stages developed faster. Thus, the neonatal ketamine injection increased the audiogenic seizure susceptibility of brain structures in KM rats.     

Does antiabsence drug ethosuximide exert antidepressant effect?

Antiabsence drug ethosuximide (300 mg/kg/day in drinking water for 17 days) produced an antidepressant effect (a decrease in immobility time in forced swimming test) only in WAG/Rij rats genetically predisposed to absence epilepsy only at age of 5 months when spike-wave discharges well pronounced. On rats without spike-wave discharges (21-day-old WAG/Rij and Wistar rats at the age of both at 21 day and 5 months), ethosuximide didn't produce the antidepressant effect but tended to increases the immobility time and significantly decreases the number of divings (active behavior oriented to escape from stressful situation). Ethosuximide didn't substantially change the anxiety level in WAG/Rij rats but significantly enhanced anxiety in 21-day-old Wistar rats. The results suggest that ethosuximide is not possessed of antidepressant potential unrelated to its suppressive effect on spike-wave discharges.     

The Responses of Neurons of the Somatosensory Cortex to Stimulation of the Posterior Thalamus (PO) in WAG/Rij Rats Genetically Predisposed to Absence Epilepsy

In genetically predisposed WAG/Rij rats and healthy Wistar rats, we studied functioning of the paralemniscal region of the thalamo-cortical system. The responses of neurons of the somatosensory cortex to single electrical stimulation of the posterior nucleus of the thalamus were recorded in two- to three-monthold rats within the period when the epileptic activity was not developed. We revealed lower number of shortterm inhibitory responses in WAG/Rij rats as compared to Wistar rats. This may create preconditions for the spreading of spike-wave activity in the somatosensory cortex, which is an electrophysiological sign of absence epilepsy.