Development and validation of the stability indicating RP-UHPLC method for the determination of the chemical purity and assay of bimatoprost.

A simple, rapid and accurate ultra-high performance liquid chromatographic (UHPLC) method with a UV detection for the determination of the chemical purity and assay of bimatoprost (BT-1) was developed. The chromatographic separation was achieved with the use of an Acquity BEH C8, 150 × 2.1 mm, 1.7 µm reversed phase analytical column. The mobile phase consisted of 0.01% H3PO4: acetonitrile (initial conditions 80 : 20, v/v) was passed through the column at the flow rate of 0.7 mL min-1. The separation was carried out in the gradient elution mode. The presented method allows to separate ten potential impurities of BT-1. The full validation according to the ICH Q2 (R1) guidelines was carried out for five of the potential impurities while limit tests were performed for four BT-1 related substances. The performed validation tests proved the suitability of the method for its intended purposes. An additional LC/MS method was utilized for the identification of the unknown impurities in bimatoprost as well as the degradation impurities generated during the forced degradation studies.

Heart rate changes in partially restrained rats during behaviorally and pharmacologically evoked emotional states.

The effect of fear and relief from fear on heart rate (HR) was studied in partially restrained adult male rats. The emotional state was influenced behaviorally with the use of Pavlovian aversive conditioning procedure, and pharmacologically by injections of the selected anxiolytics and anxiogenics. A signal of danger (DS)--light or tone, preceded tail-shock (excitatory trials), while a signal of safety (SS), respectively tone or light, overlapped last 3s of 5s DS and predicted an omission of this expected aversive event (inhibitory trials). To assess the stability of HR changes to DS and SS we analyzed whether and how the experimental conditions (modality and position of signals, the ratio of numbers of excitatory to inhibitory trials in the session) influenced HR. HR changes to DS were different in pattern, stability and direction when compared to HR changes in response to SS. Reactions to DS, although accompanied mainly by conditioned bradycardia, were not consistent and depended on experimental conditions. However, the SS always evoked conditioned tachycardia. Anxiolitycs--benzodiazepines (diazepam and midazolam) and buspirone--influenced HR in nonconsistent manner. Anxiogenics--PTZ and FG7142 were without significant effects on HR. HR could not be trusted therefore as direct index of pharmacologically induced emotional states. The results are discussed in the context of possible biobehavioral meaning of HR changes in response to danger and safety and their reliability as fear/relief correlates.

Bicuculline administration into ventromedial hypothalamus: effects on fear and regional brain monoamines and GABA concentrations in rats.

The effects of bicuculline methiodide administration into ventromedial hypothalamus (15 ng per site, bilaterally) on fear behavior and monoamines (NA, DA, 5-HT) and GABA in structures of the brain defensive system (hypothalamus, midbrain gray matter, amygdala, hippocampus and frontal cortex) were studied. Fear behavior was examined in the modified version of light-dark transition test. The time out from the illuminated compartment of chamber, the time spent there and number of returns to the illuminated compartment was measured. Additionally motor activity, i.e., number of crossings and rearings in dark as well as in the illuminated part of compartment, was registered. Blockade of GABAA receptors in the ventromedial hypothalamus resulted in increased fear behavior, i.e. decrease of time out from illuminated compartment and decrease of the time spent there. Motor behavior remained unchanged. HPLC analysis showed reduction of GABA concentration in all investigated brain structures. An increase of NA concentration in all examined structures with exception of the hypothalamus without effect on MHPG/NA was observed as well. Dopamine level remained unchanged, but DOPAC/DA ratio increased in all structures, except frontal cortex. Also HVA/DA ratio increased in the hypothalamus and midbrain. 5-HT concentration increased only in midbrain, 5-HIAA increased in midbrain and in frontal cortex, and 5-HIAA/5-HT ratio increased only in frontal cortex. These results indicate that GABA-ergic and monoaminergic systems remain in functional interactions and that these interactions may play an important role in the neurochemical regulation of fear behavior. The possible mechanism of GABA--monoaminergic interactions is discussed.

Contrasting expressions of aggressive behavior released by lesions of the central nucleus of the amygdala during wakefulness and rapid eye movement sleep without atonia in cats.

Whether damage to the central nucleus of the amygdala (Ace) contributes to the predatorylike attack sometimes observed in rapid eye movement sleep without atonia (REM-A), created in cats by bilateral pontine lesions, was examined. Such lesions eliminate REM sleep skeletal muscle atonia and release elaborate behavior. Unilateral damage to the Ace alone increased affective defensive aggressive behavior toward humans and conspecifics without altering predatory behavior in wakefulness. Pontine lesions added at loci normally not leading to aggression induced predatorylike attacks in REM-A as well as the waking affective defense. Alterations of autonomic activity, the absence of relevant environmental stimuli in REM-A, or both may explain the state-related differences.

Electrophysiological correlates of the limbic-motor interactions in various behavioral states in rats.

Depth electroencephalographic (EEG) activity was recorded from basolateral amygdala (BLA), ventral subiculum (VSB), n. accumbens (ACC) and subpallidal area (SPL) in freely moving rats, during locomotor tasks with various types of reinforcement in order to compare the strength of limbic-motor interactions in selected behavioral situations. For all EEG signals multichannel coherences (ordinary, multiple and partial) were calculated using autoregression model. Partial coherences indicate the level of synchronization between two signals, thus they were assumed to indicate the strength of direct connection between the structures from which these signals have been recorded. The partial coherences were calculated for six selected frequency bands and the strength of connections within the BLA-VSB-ACC-SPL circuit was estimated for two different behavioral situations and compared. It was found that the strength of connections is sensitive to changes in both motor and emotional aspects of behavioral situation: the strength of BLA-VSB, VSB-ACC, and ACC-SPL depended on motor demands of behavioral task; these of BLA-VSB increased in the highest frequency bands in all emotionally engaging situations when compared with well trained locomotive; the strength of ACC-SPL increased in situations when automatic stereotyped motor behavior was induced by biologically important stimuli, while it decreased or did not change in the motor tasks demanding more precise and quickly adjustable movements. The results are discussed according to the motor-limbic integration model of proposed by Mogenson and show the dynamics of its connections in relation to the motivational-emotional context of the task.

Social interactions in rats: behavioral and neurochemical alterations in DSP-4-treated rats.

Noradrenergic neurotoxin DSP-4, preceded by zimelidine to prevent serotonin depletion, was administered IP to rats behaving in a defensive-submissive manner in a resident-intruder paradigm. Computer-based ethological analysis revealed the decrease of frequency and duration of defensive episodes and marked increase of offensive aggression. This might suggest an increase of aggressiveness and therefore support the notion of an inhibitory role of the noradrenergic system in aggressive behavior independently of the model used. Dramatically changed attitude toward the partner might also result from fear reduction or inadequate responsiveness to environmental factors because DSP-4-treated rats explored more than controls in stressogenic, highly illuminated area. HPLC analysis showed significant reduction of noradrenaline (NA) concentration in amygdala, hypothalamus, hippocampus, and frontal cortex. Simultaneously there occurred a considerable decrease in dopamine (DA) and serotonin (5-HT), and their metabolite levels. This suggests an attenuated activity of the DA and 5-HT systems that we consider as an effect secondary to NA depletion, which reflects the functional interactions between DA, 5-HT, and NA systems.

DSP-4 lesion of locus coeruleus does not affect spontaneous predatory behaviour in cats.

The effect of the destruction of locus coeruleus noradrenergic (LC NA) projection on spontaneous predatory attack, predatory competition and food intake was studied in cats. Selective noradrenergic neurotoxin DSP-4 injected into LC caused 71% decrease of noradrenaline content in amygdala and 41% decrease in hypothalamus. Predatory behaviour, predatory competition as well as food intake remained unchanged. It is concluded that LC NA projection is specifically involved neither in predation nor in food intake, which is in agreement with recent electrophysiological data.

Scopolamine-induced alterations in predatory behaviour pattern in cats.

Predatory behaviour in its full pattern (i.e., following the mouse, killing it and consuming the carcass) was tested in semi-natural conditions in cats. Consumption of minced horse meat was tested as well. Centrally acting scopolamine hydrobromide injected i.p. did not suppress predatory motivation, since following the prey and killing it were preserved. The executory phase of predation (i.e., the killing grip) was severely disturbed and the consumption of the mouse as well as meat was totally inhibited. Peripherally acting scopolamine methylnitrate administered into another group of cats under the same conditions generally did not affect predatory pattern, though meat and mouse consumption was disturbed to some extent. It is concluded that central muscarinic involvement in predatory behaviour in cats is limited to sensorimotor control of jaw movements.

Changes in the aggressive behavior of cats treated with amphetamine.

Male, adult cats were tested in three different experimental conditions which might lead to aggressive display: (1) Cat-mouse interaction, (2) Predatory competition between two mice killers, (3) Conspecific interactions. DL-amphetamine in a dose of 1.5 mg/kg was administered every day for three weeks to one group of cats, whereas the other group was treated with saline. The following results were observed in amphetamine treated cats: (1) Inhibition or at least a prolonged latency of predatory attack, (2) loss of the dominant status, (3) total lack of offensive behavior and decrease of defensive postures. The results indicate that amphetamine in moderate doses acts to inhibit predatory attack and aggressive behavior in social situations.

The effect of imipramine on predatory behavior and locomotor activity in cats.

The behavior toward mouse was studied under and after chronic imipramine treatment in two groups of cats - non-killers and killers. Imiprarnine facilitated predatory behavior in the non-killers but not in the killers, which is in contrast to results obtained on rats. Imipramine produced a marked decrease of locomotor activity of non-killers tested in open field. The inhibition of locomotion did not interfere with the occurrence of killing behavior. It was concluded that imipramine selectively facilitates the neurophysiological mechanism of predatory behavior, which in cats might be connected with the reward system.

Predatory dominance and aggressive display under imipramine treatment in cats.

The effect of imipramine treatment on the aggressive display and predatory dominance was tested in 16 male cats yoked in 8 pairs. Imipramine applied chronically during 3 weeks in submissive cats produced a tendency to compete with the dominant cat in a predatory situation and an accompanying enhancement of aggressive display. Some of the treated cats gained the predatory dominance over previously dominant partners. The level of aggressiveness was not, however, directly related with the process of gaining dominance. Therefore it was concluded that imipramine enhances some mechanisms involved in dominance, not limited to aggression.

Impairment of social behavior in amygdalar cats.

Social dominance in predatory animals was tested before and after amygdala lesions. Cats after damage of dorsal part of amygdala did not show predatory behavior in group and lost their social rank in hierarchy, but in the same animals predatory attack remained unchanged in nonsocial situations. In the cat with medial amygdala lesions total abolishment of mouse-killing behavior was observed in both social and nonsocial situations. It has been concluded that amygdala plays an essential role in emotional-motivational mechanisms of predatory competitions as one of the forms of social behavior.

The effects of imipramine on socio-emotional and alimentary motivated behavior in dogs.

The effects of imipramine on spontaneous locomotor activity, alimentary motivated behavior and socio-emotional behavior were investigated in five dogs. In all dogs the main imipramine-bound effect was the decrease of locomotor activity in the open field. In four behaviorally normal dogs, performance of other tests was only slightly affected by imipramine treatment. An improvement in performance of various tasks and socio-emotional reactions was observed only in one neurotic dog.

Alimentary instrumental responses and neurological reflexes in amygdalar cats.

Lesions in ventromedial part of amygdala impaired neither alimentary instrumental reflexes nor food intake, but impaired predatory behavior. Lesions localized in the more dorsal and lateral part of the amygdala produced disorders in instrumental performance and food intake without changes in predatory attack. Both kind of lesions did not impair neurological reflexes, therefore the abolishment of predatory attack after ventromedial amygdala damage was not caused by sensory-motor disturbances. It is concluded that predatory behavior is based on specific mechanisms separate from both alimentary and instrumental ones.

Limb coordinations during locomotion in amygdalar, rubral and funicular cats.

The registration of the step phases and the relations between them for all of the four limbs were described in the amygdalar, rubral and funicular cats. The evident changes in motor behavior observed after damage of the amygdala and red nucleus did not result from coordination disorders. Conversely, the damage of the medial part of the lateral funiculus in the dorsally hemisected spinal cord disturbed the coordination between fore and hind limbs.