Enhanced neurogenesis in organotypic cultures of rat hippocampus after transient subfield-selective excitotoxic insult induced by domoic acid.

New neurons are continuously generated in the hippocampus and may play an important role in many physiological and pathological conditions. Here we present evidence of cell proliferation and neurogenesis after a selective and transient excitotoxic injury to the hippocampal cornu ammonis 1 (CA1) area induced by low concentrations of domoic acid (DOM) in rat organotypic hippocampal slice cultures (OHSC). DOM is an excitatory amino acid analog to kainic acid that acts through glutamate receptors to elicit a rapid and potent excitotoxic response. Exposure of slice cultures to varying concentrations of DOM for 24 h induced dose-dependent neuronal toxicity that was independent of activation of classic apoptotic markers. Treatment with 2 µM DOM for 24 h caused a selective yet transient neurotoxic injury in the CA1 subfield of the hippocampus that appeared recovered after 7 days of incubation in a DOM-free medium and showed significant microgliosis but no sign of astrogliosis. The DOM insult (2 µM, 24 h) resulted in a significant upregulation of cell proliferation, as assessed by 5-bromo-2-deoxyuridine (BrdU) incorporation, and a concurrent increase of the neuronal precursor cell marker doublecortin (DCX) within the subgranular zone of the dentate gyrus and area CA1. Neurogenesis occurred primarily during the first week after termination of the DOM exposure. Our study shows that exposure of OHSC to concentrations of DOM below those required to induce permanent neurotoxicity can induce proliferation and differentiation of neural progenitor cells that may contribute to recovery from mild injury and to develop abnormal circuits relevant to disease.

Influence of diurnal phase on startle response in adult rats exposed to dexamethasone in utero.

Depression and pathological anxiety disorders are among the most prevalent neurological diseases in the world and can be precipitated and exacerbated by stress. Prenatal stress alters both behavioral and endocrine responses to stressful stimuli in later life. We have previously observed increased basal acoustic startle response (ASR) in Wistar rats exposed to stress or dexamethasone (DEX) in utero when tested during the light phase of the circadian rhythm, and decreased prepulse inhibition (PPI) in similar animals tested during the dark phase of the cycle. We speculated that this observation of increased basal startle might be influenced by diurnal phase. In the present study, adult female Sprague Dawley rats, stressed prenatally with DEX (200 µg/kg, gestational days 14-21) and postnatally by blood sampling under restraint, were tested for the ASR during both circadian phases (light and dark). Basal startle was increased in animals tested both during the light and the dark phases of the cycle. We hereby replicated our earlier findings in a new strain and laboratory, thus strengthening the validity of our model regarding prenatal stress effects on ASR in female offspring. Our results indicate that observation of increased basal ASR is not solely dependent on diurnal phase. We found no difference in hippocampal glucocorticoid and mineral corticoid receptor expression between groups.

Peroxisome proliferator-activated receptor-γ (PPAR-γ) activation confers functional neuroprotection in global ischemia.

The neuroprotective effect of rosiglitazone, a peroxisome proliferator-activated receptor γ (PPARγ) agonist, has been investigated using both in vivo and in vitro models of global ischemia in CD1 mice. Behavioral tests were carried out prior to and at various times (up to 14 days) subsequent to bilateral common carotid artery occlusion followed by reperfusion. Mice at each time point were euthanized under anesthesia and the brain was removed, serially sliced and stained with 1% triphenyltetrazolium (TTC) to quantify infarct size. Administration of rosiglitazone (5 or 10 mg/kg, i.p.) 10 min prior to occlusion significantly reduced the postsurgical mortality rate (10-11 vs. 36%, P < 0.05). The higher dose of rosiglitazone (10 mg/kg) also significantly reduced the mean area of brain infarct at 1, 3, 7 and 14 days post-ischemia, reduced post-occlusion deficits in limb grasping and forelimb placing at various time points, and reduced total nitrite concentration in serum and brain homogenate at day 7 post-occlusion. To model global ischemia in vitro, coronal brain slices were incubated in oxygenated artificial cerebrospinal fluid (ACSF) in the presence of either glutamate (1 mM) or hydrogen peroxide (H(2)O(2)) (5 µM) for 30 min. Both H(2)O(2) and glutamate caused significant tissue damage, and co-incubation with rosiglitazone (5 µM) significantly reduced H(2)O(2)-induced damage but did not significantly reduce glutamate-induced brain damage in this model. Our observations provide further evidence for a neuroprotective effect of rosiglitazone in rodent models of ischemia.

Neonatal exposure to low-dose domoic acid lowers seizure threshold in adult rats.

Exposing Sprague-Dawley rat pups to very low, sub-convulsant doses of domoic acid (DOM) during perinatal development has been previously shown to result in seizure-like activity in adulthood similar to partial complex epilepsy in humans, and to produce cellular and molecular changes in the dentate gyrus and area CA-3 of the hippocampus. To further these investigations we recorded electroencephalographical and behavioural activity in DOM and control rats following a normally sub-convulsant dose (25 mg/kg) of pentylenetetrazol. During this exposure, 50% of DOM-treated rats experienced a Stage V (tonic-clonic) seizure (X(2)((1))=5.33, P=0.021), indicating a lowering of generalized seizure threshold in these animals. In a separate experiment we explored focal seizure (afterdischarge) threshold as well as seizure propagation rates in treated rats, using a 25 consecutive day standard amygdala kindling paradigm. We report that the afterdischarge threshold for DOM-treated rats was significantly lower than controls (F((1,27))=7.117, P=0.013). No difference between groups was found in seizure progression as measured by afterdischarge duration, latency to first Stage V seizure, or latency to reach a fully kindled state (defined as five consecutive Stage V seizures). Timm staining to assess mossy fibre sprouting (MFS) in the hippocampus revealed a significant MFS increase relative to sham at the ventral level in both left and right inner molecular layer of the dentate gyrus for all DOM-treated animals, as well as in the dorsal stratum oriens of CA3 contralateral to electrode placement, and these increases were further enhanced by the kindling procedure. We conclude that perinatal exposure to subconvulsive doses of DOM results in permanent changes in neuronal excitability in the adult rat, as demonstrated by a lowering of both generalized seizure and focal afterdischarge threshold, and produces increased MFS following kindling.

Reductions in paradoxical sleep time in adult rats treated neonatally with low dose domoic acid.

One hallmark of neurological dysfunction is a reduction in paradoxical sleep (PS) time. To determine if adult rats treated neonatally with low dose domoic acid have altered sleep patterns, a home cage analysis of electroencephalogram (EEG) waveforms was performed using radio telemetry. Domoate treated rats spent significantly less time in PS than controls during daytime hours even though they spent the same total amount of time sleeping, and showed no difference in stage shifts into the PS stage.

Gender-based changes in cognition and emotionality in a new rat model of epilepsy.

Epilepsy research relies heavily on animal models that mimic some, or all, of the clinical symptoms observed. We have previously described a new developmental rat model of epilepsy that demonstrates both behavioural seizures and changes in hippocampal morphology. In the current study we investigated whether these rats also show changes in cognitive performance as measured using the Morris water maze task, and emotionality as measured using the Elevated plus maze task. In the water maze, significant differences between male and female rats were found in several performance variables regardless of treatment. In addition, female but not male rats, treated neonatally with domoic acid had significant impairments in learning new platform locations in the water maze. In the elevated plus maze, a significant proportion of female rats spent more time in the open arm of the maze following prior exposure to the maze whereas this effect was not seen in male rats. We conclude that perinatal treatment with low doses of domoic acid results in significant gender-based changes in cognition and emotionality in adult rats.

NMDA receptor involvement in the effects of low dose domoic acid in neonatal rats.

We have previously reported that neonatal rats display enhanced sensitivity to domoic acid relative to adults, and that perinatal injections of low doses of domoic acid alter early associational learning in the newborn rat. The current study was designed to further investigate the effects of low dose domoic acid on neonatal odour conditioning and to determine if the observed effects are due in part to an action on NMDA receptors. Groups of rat pups were conditioned to a novel odour on postnatal day (PND) 8, injected with 20 microg/kg domoic acid either alone, or in combination with the NMDA antagonist CPP (or appropriate controls), daily from day 8-14, re-exposed to the conditioning odour or a novel odour on day 9, and tested for odour preference on day 13 using a standard 3-choice paradigm. Results indicated that rats treated with domoic acid spent significantly more time over the conditioning odour than did saline-treated rats when tested on PND 13. This effect was antagonized by concomitant injection of CPP, indicating an involvement of NMDA receptors in the actions of DOM in this paradigm. Rats injected with either saline or CPP alone showed the opposite effect, i.e. a preference for the alternate odour. The results indicate that a very low dose of DOM produces a conditioned odour preference in neonatal rats and that this effect is due in part to NMDA receptor involvement, thereby emphasizing a role for both kainate and NMDA glutamate receptors in implicit memory.

Low doses of domoic acid during postnatal development produce permanent changes in rat behaviour and hippocampal morphology.

It is well established that the developing brain is a highly dynamic environment that is susceptible to toxicity produced by a number of pharmacological, chemical and environmental insults. We report herein on permanent behavioural and morphological changes produced by exposing newborn rats to very low (subconvulsive) doses of kainate receptor agonists during a critical window of brain development. Daily treatment of SD rat pups with either 5 or 20 microg/kg of domoic acid (DOM) from postnatal day 8-14 resulted in a permanent and reproducible seizure-like syndrome when animals were exposed to different tests of spatial cognition as adults. Similar results were obtained when animals were treated with equi-efficacious doses of kainic acid (KA; 25 or 100 microg/kg). Treated rats had significant increases in hippocampal mossy fiber staining and reductions in hippocampal cell counts consistent with effects seen in adult rats following acute injections of high doses of kainic acid. In situ hybridization also revealed an elevation in hippocampal brain derived neurotrophic factor (BDNF) mRNA in region CA1 without a corresponding increase in neuropeptide Y (NPY) mRNA. These results provide evidence of long-lasting behavioural and histochemical consequences arising from relatively subtle changes in glutamatergic activity during development, that may be relevant to understanding the aetiology of seizure disorders and other forms of neurological disease.

Comparison of the in vitro and in vivo neurotoxicity of three new sources of kainic acid.

Historically, all commercially available kainic acid has been derived from a single biological source using a consistent method of extraction and purification. That source became unavailable in 1995. Recently, three new commercial suppliers of kainic acid have made the product available, but the source of the material and the purification processes used differ. Our objective was to systematically compare the response produced by each of these new sources of kainic acid using three established neurobiological techniques: neuronal cell culture, hippocampal slice electrophysiology, and whole animal behavioural toxicity. Results in all three systems indicated no overall differences between the three formulations, although studies in both cerebellar neuron cultures and whole animal toxicity testing in mice, revealed some significant differences that may imply subtle differences in receptor selectivity and/or potency. We conclude that all three sources of kainic acid are viable alternatives to traditional kainate but they may not be identical. Until further information becomes available researchers may want to avoid using the three formulations interchangeably, and take note of the source of kainic acid when evaluating literature describing results from other laboratories.

Comparative behavioural toxicity of domoic acid and kainic acid in neonatal rats.

Cumulative behavioural toxicity was measured in groups of male and female rat pups (n=6/sex) at different stages of postnatal development. Dose-response curves (DRCs) for toxicity produced by domoic acid (DOM) were generated using animals on postnatal days (PND) 0, 5, 14, and 22, using a behavioural rating scale. In a subsequent experiment, DRCs for toxicity generated by either DOM or kainic acid were produced in rats at PND 8 and 14 for comparison between the two toxins. DOM was found to be a very potent neurotoxin in newborn rats and the potency of DOM progressively decreased with increasing age (interpolated ED(50)=0.12, 0.15, 0.30, and 1.06 mg/kg at PND 0, 5, 14, and 22, respectively). In addition, the patterns of behavioural expression were found to differ with age. Comparisons between DOM and kainic acid revealed that DOM was approximately six-fold more potent than kainate at both PND 8 and PND 14 and that both toxins were approximately two-fold less potent in PND 14 rats, compared to PND 8. This implies that the mechanism(s) responsible for reduced potency is/are similar between the two compounds. Consistent with previous reports, however, there were both similarities and differences in the observed patterns of behavioural toxicity produced by the two toxins at both ages.

Use of osmotic minipumps for sustained drug delivery in rat pups: effects on physical and neurobehavioural development.

Osmotic minipumps are often used as an alternative to repetitive injections for prolonged drug delivery in adult rats. The appropriateness of using this technology for sustained drug delivery in newborn rats, however, has not been validated. Our objective was to determine if implantation of osmotic minipumps, and the associated surgical stress, during a critical developmental period, affects early development and subsequent behaviour. SD rat pups were assigned to control, minipump, or sham surgery treatment conditions (n=12/group). On P8, pups were briefly anaesthetised with isoflurane in oxygen, and Alzet 1007D osmotic minipumps, loaded with normal saline, were aseptically implanted (removed on P17). Sham-treated rats received identical treatment (with the exception of pump placement), while control pups were left undisturbed. Development was examined daily using a standard test battery (P9-P21), and learning and memory in pups was assessed in a T-maze (P15, P17 and P19). Weight (P27 and P72), open-field (P25, P26 and P27) and novel water maze performance (P60-P72) were examined in the resulting adult. With the exception of a transient decrease in weight gain, pump-treated animals did not differ from either sham or control rats, on any pre- or postweaning assessment. Based on these results we conclude that the use of osmotic minipumps in rat pups is a viable alternative to repeated injections for sustained drug delivery.

Synergism between NMDA and domoic acid in a murine model of behavioural neurotoxicity.

We have examined the behavioural neurotoxicity of domoic acid (DOM) and kainic acid (KA) in mice following administration of ligands active at the N-methyl-d-aspartate (NMDA) receptor. Groups of female CD-1 mice (n=4) were injected i.p. with saline or one of three doses of either DOM or KA. Doses of DOM and KA were selected from the steep portion of the respective dose response curves and were equitoxic when compared between the two ligands. Toxicity was recorded as both total cumulative toxicity over 60 min according to a previously validated 7 point rating scale, and as the latency to the onset of tremors and/or convulsions. Five minutes prior to administration of either agonist mice were injected with either saline, NMDA (40 mg/kg) or a combination of NMDA and 15 mg/kg CPP (3-[2-carboxypiperazine-4-yl]propyl-1-phosphonic acid). Neither NMDA nor CPP at these doses produced significant changes from baseline responding when injected prior to saline. Injection of NMDA prior to DOM, however, resulted in significantly increased cumulative toxicity and significantly reduced latencies to seizures at the two highest doses of DOM (3.75 and 5.0 mg/kg). NMDA-induced potentiation of DOM toxicity was completely antagonized by co-administration of CPP. In contrast, injection of NMDA prior to KA did not result in significant changes in KA toxicity at any of the doses tested using either index of behavioural toxicity. These results confirm previous reports of synergism between DOM and ligands acting at the NMDA receptor in isolated neurons, and provide further evidence of pharmacological dissociation of the actions of DOM and KA in vivo.

Development of an injectable sustained-release formulation of morphine: antinociceptive properties in rats.

The antinociceptive actions of morphine incorporated into an injectable chitosan-based gel were investigated in rats. Subcutaneous administration of 4.8 mg/kg morphine sulphate in a gel composed of N,O-carboxymethylchitosan (NOCC) and chitosan resulted in significant antinociception within 10 min that was maximal at 60 min and persisted for 6 h. In contrast, the same dose of morphine sulphate injected in sterile saline produced maximal responses at 30 min but only persisted for 2 h. NOCC/chitosan gel was easily injectable using a 22 guage needle and appears stable in long-term storage. No local or systemic adverse effects other than morphine-induced sedation were observed either at the time of injection or during the subsequent 48 h. We conclude that gels composed of chitosan and chitosan derivatives are effective matrices for sustained-release formulations of opioid analgesics capable of providing long-lasting antinociception.

Pharmacokinetics of an injectable sustained-release formulation of morphine for use in dogs.

This study investigated the pharmacokinetics of morphine sulphate in an injectable chitosan-based gel. Gels were made from a combination of N-O-carboxymethylchitosan (NOCC) and chitosan and were easily injectable via a 22 gauge needle and appeared stable during long-term storage. Groups of six beagles were injected subcutaneously (s.c.) with 1.2 mg/kg morphine sulphate, either in sterile saline or in sterilized gels, and serial blood samples were withdrawn via a jugular catheter and later analysed for morphine concentrations using radioimmunoassay. Data were analysed according to non-compartmental pharmacokinetics. NOCC-based gels resulted in significantly lower serum morphine concentrations at 10 and 30 min following injection but significantly higher concentrations at all points from 120 to 480 min post-injection. Dogs receiving morphine gel exhibited equivalent or lesser variability in serum morphine concentrations than dogs receiving conventional morphine sulphate. Pharmacokinetic analysis revealed that morphine release from the gel matrix was significantly prolonged but fully bioavailable. There were no significant differences in either distribution (Vd) or terminal elimination (t 1/2). Dogs experienced no adverse effects other than those normally associated with morphine administration at the time of injection but all dogs receiving the gel presented with an undefined stiffness the next day that resolved spontaneously within 48 h. We conclude that carboxymethylchitosan-based gels hold considerable promise for the development of injectable sustained-release formulations of opioid analgesics.

Pharmacokinetics of oral morphine sulfate in dogs: a comparison of sustained release and conventional formulations.

The pharmacokinetics and bioavailability (F) of single dose sustained release morphine sulfate (OSRMS) and nonsustained release morphine sulfate (NSRMS) were compared to each other and to a bolus injection of morphine sulfate (MS) intravenously (i.v.) in dogs. Beagles (n = 6) were randomly assigned to 3 treatment groups: namely, OSRMS 15 mg orally, NSRMS 15 mg orally, and 15 mg i.v. Serum samples were drawn at intervals up to 480 min following oral and 420 min following i.v. administration. Serum was analysed for morphine concentration using a radioimmunoassay. Data were analysed using non-compartmental pharmacokinetics. The only statistically significant difference between OSRMS and NSRMS was maximum serum concentration (Cmax). There were trends toward longer time to maximum serum concentration (Tmax) and longer mean absorption time (MAT) for OSRMS when compared to NSRMS, but the differences were not statistically significant (P < 0.05). Pharmacokinetic parameters for both oral formulations exhibited large variability in the rate of absorption of MS from the gastrointestinal tract. Bioavailability of both OSRMS and NSRMS was low (15%-17%). As expected, the area under the concentration vs time curve (AUC) and Cmax for the i.v. data was significantly greater than for both oral groups, and Tmax and mean residence time (MRT) were significantly less following i.v. administration. There were no statistically significant differences among the 3 treatment groups for apparent volume of distribution at steady state (Vdss) or elimination parameters. The OSRMS formulation used in this study provided equivalent bioavailability to NSRMS in dogs, accompanied by large individual variability in drug absorption. It also did not appear that the sustained release formulation provided sufficiently prolonged release of morphine sulfate from the tablet matrix in dogs to allow prolonged dosing intervals compared to NSRMS.

Selective reduction in domoic acid toxicity in vivo by a novel non-N-methyl-D-aspartate receptor antagonist.

Our objective was to characterize the neurotoxic actions of systemically administered domoic acid on different excitatory amino acid receptors, and to compare the receptor selectivity of domoate with the related compound kainic acid. Groups of mice were injected with various ligands selective for N-methyl-D-aspartate (NMDA) and 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid-kainate (AMPA/kainate) receptors prior to injection of equitoxic doses of domoic acid or kainic acid. Domoic acid toxicity was not significantly altered by pretreatment with any NMDA receptor selective antagonists, with the exception of 3-(2-carboxypiperazine-4-yl)propyl-1 -phosphonic acid. Consistent with its characterization as an AMPA/kainate agonist, domoate toxicity was significantly antagonized by all non-NMDA receptor antagonists tested. Non-NMDA receptor antagonists that do not distinguish between high- and low-affinity [3H]kainic acid binding (i.e., quinoxalinediones) were equally effective at reducing domoic acid and kainic acid toxicity. However, the novel isatinoxime NS-102, which has been shown to interact selectively with low-affinity [3H]kainic acid binding sites, produced a selective dose-related antagonism of domoic acid toxicity relative to kainic acid. NS-102 produced significant reductions in overall toxicity, onset of motor seizures, and hippocampal CA3 cell damage induced by domoic acid at NS-102 doses that did not antagonize kainic acid induced toxicity. We conclude that domoic acid toxicity in vivo is mediated largely by a subclass of non-NMDA receptors that are selectively antagonized by NS-102.

Pharmacokinetics of parenteral and oral sustained-release morphine sulphate in dogs.

The pharmacokinetics of single-dose morphine sulphate (MS) administered intravenously (i.v.) and intramuscularly (i.m.) and of oral sustained-release morphine sulphate (OSRMS) were studied in dogs. Beagles (n = 6) were randomly assigned to six treatment groups using a Latin square design. Treatments included MS 0.5 and 0.8 mg/kg i.v. and i.m. and OSRMS 15 and 30 mg orally (p.o). Serum samples were drawn at intervals up to 420 min following parenteral MS and 720 min following OSRMS. Serum was analysed for morphine concentration using a radioimmunoassay. Pharmacokinetic analysis of the results revealed that MS was eliminated by a first-order process best described by a two-compartment model. For i.v. and i.m. data there were no statistically significant differences (P < 0.05) between steady-state volume of distribution, half-life of elimination and plasma clearance. As expected, area under the concentration vs. time curve (AUC) was significantly greater for the 0.8 mg/kg dosage for i.v. and i.m. routes, and time to maximum serum concentration was significantly longer following i.m. administration. For OSRMS there were no significant differences between dosage for any parameter (AUC, Cmax, tmax, t1/2, F) and prolonged absorption of the drug occurred over approximately 6 h. Bioavailability (F) for both oral dosages was approximately 20%. The i.m. route is an effective method for rapid and complete delivery of MS to dogs. OSRMS may be useful in the provision of long-term analgesic therapy in dogs, but further work is required to verify the safety and effectiveness of this preparation.

Rapid development of tolerance to morphine in the formalin test.

Previous reports have suggested that tolerance to the antinociceptive effects of morphine does not develop in the formalin test. To re-examine these findings, morphine was administered via multiple injections or continuous infusion from 0 to 4 days prior to testing with either the formalin or tail-flick test. Following twice daily injections (5.0 mg kg-1 s.c.), significant tolerance developed within 2 days with the formalin test but not until day 4 with the tail-flick test. Significant tolerance was noted with both tests 4 days following the implantation of osmotic mini-pumps (10 mg kg-1 day-1, s.c.). We conclude that in mice, tolerance to the analgesic effects of morphine develops rapidly with the formalin test.