Behavioral sensitization and tolerance induced by repeated treatment with ketamine enantiomers in male Wistar rats.

Ketamine has gained significant attention as a fast-acting antidepressant. However, ketamine is also associated with undesirable side effects. In our preclinical study, we explored the behavioral effects of ketamine enantiomers at subanesthetic doses. During repeated intermittent treatment, we examined locomotor stimulation and sensitization, ataxia, and expression of natural behaviors (grooming and rearing). Male Wistar rats were subcutaneously treated repeatedly with either 5 mg/kg of R-ketamine or S-ketamine, 15 mg/kg of R-ketamine, S-ketamine or racemic ketamine, 30 mg/kg of racemic ketamine or saline every third day for three weeks (seven treatments overall). After the first treatment, only 15 mg/kg of S-ketamine induced locomotor stimulation, and both 15 mg/kg of S-ketamine and 30 mg/kg of racemic ketamine induced ataxia. Upon repeated administration, doses of 15 mg/kg of R-ketamine, S-ketamine, and racemic ketamine, as well as 30 mg/kg of racemic ketamine, stimulated locomotion. 15 mg/kg of R-ketamine, S-ketamine, and racemic ketamine additionally resulted in locomotor sensitization. The last administration of 15 mg/kg of S-ketamine, 15 mg/kg of racemic ketamine, and 30 mg/kg of racemic ketamine resulted in ataxia. In the case of 15 mg/kg of S-ketamine, ataxic effects were significantly weaker in comparison to the effects from the first administration, indicating tolerance. Natural behaviors were attenuated after 5 and 15 mg/kg of S-ketamine and 15 and 30 mg/kg of racemic ketamine. Neither of the R-ketamine doses produced such an effect. We conclude that S-ketamine has a stronger behavioral effect than R-ketamine.

Increased pituitary adenylate cyclase-activating polypeptide in the central bed nucleus of the stria terminalis in mood disorders in men.

The mood disorders major depressive disorder (MDD) and bipolar disorder (BD) are highly prevalent worldwide. Women are more vulnerable to these psychopathologies than men. The bed nucleus of the stria terminalis (BNST), the amygdala, and the hypothalamus are the crucial interconnected structures involved in the stress response. In mood disorders, stress systems in the brain are put into a higher gear. The BNST is implicated in mood, anxiety, and depression. The stress-related neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is highly abundant in the central BNST (cBNST). In this study, we investigated alterations in PACAP in the cBNST of patients with mood disorders. Immunohistochemical (IHC) staining of PACAP and in situ hybridization (ISH) of PACAP mRNA were performed on the cBNST of post-mortem human brain samples. Quantitative IHC revealed elevated PACAP levels in the cBNST in both mood disorders, MDD and BD, but only in men, not in women. The PACAP ISH was negative, indicating that PACAP is not produced in the cBNST. The results support the possibility that PACAP innervation of the cBNST plays a role in mood disorder pathophysiology in men.

Alterations in pituitary adenylate cyclase-activating polypeptide in major depressive disorder, bipolar disorder, and comorbid depression in Alzheimer's disease in the human hypothalamus and prefrontal cortex.

BACKGROUND: Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) is involved in the stress response and may play a key role in mood disorders, but no information is available on PACAP for the human brain in relation to mood disorders. METHODS: PACAP-peptide levels were determined in a major stress-response site, the hypothalamic paraventricular nucleus (PVN), of people with major depressive disorder (MDD), bipolar disorder (BD) and of a unique cohort of Alzheimer's disease (AD) patients with and without depression, all with matched controls. The expression of PACAP-(Adcyap1mRNA) and PACAP-receptors was determined in the MDD and BD patients by qPCR in presumed target sites of PACAP in stress-related disorders, the dorsolateral prefrontal cortex (DLPFC) and anterior cingulate cortex (ACC). RESULTS: PACAP cell bodies and/or fibres were localised throughout the hypothalamus with differences between immunocytochemistry and in situ hybridisation. In the controls, PACAP-immunoreactivity-(ir) in the PVN was higher in women than in men. PVN-PACAP-ir was higher in male BD compared to the matched male controls. In all AD patients, the PVN-PACAP-ir was lower compared to the controls, but higher in AD depressed patients compared to those without depression. There was a significant positive correlation between the Cornell depression score and PVN-PACAP-ir in all AD patients combined. In the ACC and DLPFC, alterations in mRNA expression of PACAP and its receptors were associated with mood disorders in a differential way depending on the type of mood disorder, suicide, and psychotic features. CONCLUSION: The results support the possibility that PACAP plays a role in mood disorder pathophysiology.

Pseudo-irreversible butyrylcholinesterase inhibitors: Structure-activity relationships, computational and crystallographic study of the N-dialkyl O-arylcarbamate warhead.

Alongside reversible butyrylcholinesterase inhibitors, a plethora of covalent butyrylcholinesterase inhibitors have been reported in the literature, typically pseudo-irreversible carbamates. For these latter, however, most cases lack full confirmation of their covalent mode of action. Additionally, the available reports regarding the structure-activity relationships of the O-arylcarbamate warhead are incomplete. Therefore, a follow-up on a series of pseudo-irreversible covalent carbamate human butyrylcholinesterase inhibitors and the structure-activity relationships of the N-dialkyl O-arylcarbamate warhead are presented in this study. The covalent mechanism of binding was tested by IC50 time-dependency profiles, and sequentially and increasingly confirmed by kinetic analysis, whole protein LC-MS, and crystallographic analysis. Computational studies provided valuable insights into steric constraints and identified problematic, bulky carbamate warheads that cannot reach and carbamoylate the catalytic Ser198. Quantum mechanical calculations provided further evidence that steric effects appear to be a key factor in determining the covalent binding behaviour of these carbamate cholinesterase inhibitors and their duration of action. Additionally, the introduction of a clickable terminal alkyne moiety into one of the carbamate N-substituents and in situ derivatisation with azide-containing fluorophore enabled fluorescent labelling of plasma human butyrylcholinesterase. This proof-of-concept study highlights the potential of this novel approach and for these compounds to be further developed as clickable molecular probes for investigating tissue localisation and activity of cholinesterases.

Rat Group IIA Secreted Phospholipase A2 Binds to Cytochrome c Oxidase and Inhibits Its Activity: A Possible Episode in the Development of Alzheimer's Disease.

Alzheimer's disease (AD), a progressive form of dementia, is characterized by the increased expression of secreted phospholipase A2 group IIA (GIIA) in the affected tissue and the dysfunction of neuronal mitochondria, similar to that induced by an orthologous GIIA from snake venom, ß-neurotoxic ammodytoxin (Atx), in the motor neurons. To advance our knowledge about the role of GIIA in AD, we studied the effect of rat GIIA on the neuronal mitochondria and compared it with that of the Atx. We produced recombinant rat GIIA (rGIIA) and its enzymatically inactive mutant, rGIIA(D49S), and demonstrated that they interact with the subunit II of cytochrome c oxidase (CCOX-II) as Atx. rGIIA and rGIIA(D49S) bound to this essential constituent of the respiratory chain complex with an approximately 100-fold lower affinity than Atx; nevertheless, both rGIIA molecules potently inhibited the CCOX activity in the isolated rat mitochondria. Like Atx, rGIIA was able to reach the mitochondria in the PC12 cells from the extracellular space, independent of its enzymatic activity. Consistently, the inhibition of the CCOX activity in the intact PC12 cells and in the rat's brain tissue sections was clearly demonstrated using rGIIA(D49S). Our results show that the effects of mammalian and snake venom ß-neurotoxic GIIA on the neuronal mitochondria have similar molecular backgrounds. They suggest that the elevated extracellular concentration of GIIA in the AD tissue drives the translocation of this enzyme into local neurons and their mitochondria to inhibit the activity of the CCOX in the respiratory chain. Consequently, the process of oxidative phosphorylation in the neurons is attenuated, eventually leading to their degeneration. Atx was thus revealed as a valuable molecular tool for further investigations of the role of GIIA in AD.

Modulation by Estradiol of L-Dopa-Induced Dyskinesia in a Rat Model of Post-Menopausal Hemiparkinsonism.

Treatment with levodopa (L-dopa) in Parkinson's disease (PD) leads to involuntary movements termed L-dopa-induced dyskinesia (LID). There are contradictory data about the influence of hormone therapy in female PD patients with LID and of 17-ß-estradiol (E2) on animal correlates of LID-abnormal involuntary movements (AIMs). Our aim was to characterize the influence of E2 on motor impairment and AIMs in ovariectomized 6-hydroxydopamine (6-OHDA) rat model of PD. Half of the rats received empty and the other half implants filled with E2. Following the 6-OHDA surgery, the rats received daily treatment with either L-dopa or saline for 16 days. They were assessed for AIMs, contralateral rotations, and FAS. In the L-dopa-treated rats, E2 intensified and prolonged AIMs and contralateral rotations. On the other hand, it had no effect on motor impairment. Postmortem tyrosine hydroxylase immunostaining revealed an almost complete unilateral lesion of nigrostriatal dopaminergic neurons. E2 partially prevented the upregulation of striatal ΔFosB caused by dopamine depletion. L-dopa potentiated the upregulation of ΔFosB within the dopamine-depleted striatum and this effect was further enhanced by E2. We speculate that the potentiating effects of E2 on AIMs and on contralateral rotations could be explained by the molecular adaptations within the striatal medium spiny neurons of the direct and indirect striatofugal pathways.

Effects of L-dopa on expression of prolactin and synaptotagmin IV in 17-beta-estradiol-induced prolactinomas of ovariectomized hemiparkinsonian rats.

Parkinson's disease (PD) is a long-term degenerative disorder of the central nervous system that mainly affects the motor system. Dopamine precursor levodopa (L-dopa) is used as the first-line treatment for PD. Evidence suggests neuroprotective effects of estrogens in PD. Since both 17b-estradiol (E2) and L-dopa act as regulators of prolactin (PRL) secretion from the pituitary gland, we investigated their effect on the expression of PRL in prolactinomas that developed in ovariectomized hemiparkinsonian rats treated with E2. We also investigated the effect of E2 and L-dopa on the expression of synaptotagmin IV (Syt IV), an immediate early gene whose product is abundant in the pituitary gland and was found to be highly co-expressed with PRL in lactotrophs (>90%). The hemiparkinsonian rat model was obtained by unilateral lesioning of dopaminergic nigrostriatal neurons. Rats received silastic tubing implants with E2 and were treated with L-dopa. Enzyme-linked immunosorbent assay and immunohistochemistry were used to assess the serum concentrations of PRL and E2 and expression of PRL and Syt IV in the tissue of adenohypophysis, respectively. We found that high levels of serum E2 were associated with the upregulation of Syt IV and PRL in PRL-ir cells, while treatment with L-dopa decreased the size of prolactinomas and downregulated Syt IV but had no effect on PRL expression or serum concentrations.

Development of potent reversible selective inhibitors of butyrylcholinesterase as fluorescent probes.

Brain butyrylcholinesterase (BChE) is an attractive target for drugs designed for the treatment of Alzheimer's disease (AD) in its advanced stages. It also potentially represents a biomarker for progression of this disease. Based on the crystal structure of previously described highly potent, reversible, and selective BChE inhibitors, we have developed the fluorescent probes that are selective towards human BChE. The most promising probes also maintain their inhibition of BChE in the low nanomolar range with high selectivity over acetylcholinesterase. Kinetic studies of probes reveal a reversible mixed inhibition mechanism, with binding of these fluorescent probes to both the free and acylated enzyme. Probes show environment-sensitive emission, and additionally, one of them also shows significant enhancement of fluorescence intensity upon binding to the active site of BChE. Finally, the crystal structures of probes in complex with human BChE are reported, which offer an excellent base for further development of this library of compounds.

On the spot: utilization of directional cues in vibrational communication of a stink bug.

Although vibrational signalling is among the most ancient and common forms of communication, many fundamental aspects of this communication channel are still poorly understood. Here, we studied mechanisms underlying orientation towards the source of vibrational signals in the stink bug Nezara viridula (Hemiptera, Pentatomidae), where female vibrational song enables male to locate her on the bean plant. At the junction between the main stem and the leaf stalks, male placed his legs on different sides of the branching and orientation at the branching point was not random. Analyses of signal transmission revealed that only a time delay between the arrival of vibrational wave to receptors located in the legs stretched across the branching was a reliable directional cue underlying orientation, since, unexpectedly, the signal amplitude at the branching point was often higher on the stalk away from the female. The plant and the position of the vibrational source on the plant were the most important factors influencing the unpredictability of the amplitude cue. Determined time delays as short as 0.5 ms resulted in marked changes in interneuron activity and the decision model suggests that the behavioural threshold is in the range between 0.3 and 0.5 ms.

Descending brain neurons in the cricket Gryllus bimaculatus (de Geer): auditory responses and impact on walking.

The activity of four types of sound-sensitive descending brain neurons in the cricket Gryllus bimaculatus was recorded intracellularly while animals were standing or walking on an open-loop trackball system. In a neuron with a contralaterally descending axon, the male calling song elicited responses that copied the pulse pattern of the song during standing and walking. The accuracy of pulse copying increased during walking. Neurons with ipsilaterally descending axons responded weakly to sound only during standing. The responses were mainly to the first pulse of each chirp, whereas the complete pulse pattern of a chirp was not copied. During walking the auditory responses were suppressed in these neurons. The spiking activity of all four neuron types was significantly correlated to forward walking velocity, indicating their relevance for walking. Additionally, injection of depolarizing current elicited walking and/or steering in three of four neuron types described. In none of the neurons was the spiking activity both sufficient and necessary to elicit and maintain walking behaviour. Some neurons showed arborisations in the lateral accessory lobes, pointing to the relevance of this brain region for cricket audition and descending motor control.

Temporal processing of vibratory communication signals at the level of ascending interneurons in Nezara viridula (Hemiptera: Pentatomidae).

During mating, males and females of N. viridula (Heteroptera: Pentatomidae) produce sex- and species-specific calling and courtship substrate-borne vibratory signals, grouped into songs. Recognition and localization of these signals are fundamental for successful mating. The recognition is mainly based on the temporal pattern, i.e. the amplitude modulation, while the frequency spectrum of the signals usually only plays a minor role. We examined the temporal selectivity for vibratory signals in four types of ascending vibratory interneurons in N. viridula. Using intracellular recording and labelling technique, we analyzed the neurons' responses to 30 pulse duration/interval duration (PD/ID) combinations. Two response arrays were created for each neuron type, showing the intensity of the responses either as time-averaged spike counts or as peak instantaneous spike rates. The mean spike rate response arrays showed preference of the neurons for short PDs (below 600 ms) and no selectivity towards interval duration; while the peak spike rate response arrays exhibited either short PD/long ID selectivity or no selectivity at all. The long PD/short ID combinations elicited the weakest responses in all neurons tested. No response arrays showed the receiver preference for either constant period or duty cycle. The vibratory song pattern selectivity matched the PD of N. viridula male vibratory signals, thus pointing to temporal filtering for the conspecific vibratory signals already at level of the ascending interneurons. In some neurons the responses elicited by the vibratory stimuli were followed by distinct, regular oscillations of the membrane potential. The distance between the oscillation peaks matched the temporal structure of the male calling song, indicating a possible resonance based mechanism for signal recognition.

Morphology and physiology of vibratory interneurons in the thoracic ganglia of the southern green stinkbug Nezara viridula (L.).

The central processing mechanisms of vibratory signals in small plant-dwelling insects that rely primarily on substrate-borne vibratory communication are still largely unknown. To elucidate the neural mechanisms involved in vibratory signaling, the vibration-sensitive interneurons in thoracic ganglia of the southern green stinkbug, Nezara viridula, were investigated electrophysiologically by single-cell recordings and staining. Ten types of interneurons were described and divided into four categories, based on their gross morphology. The cell body of the L-shaped CG-AC neurons is located in the metathoracic neuromere of the central ganglion, and the axon ascends contralaterally. This group comprises five types of neurons differing in their fine structure and functional properties. CG-AB neurons are dorsal unpaired median (DUM) neurons with cell bodies in the mesothoracic neuromere of the central ganglion and two axons that ascend bilaterally into the prothoracic ganglion. Group CG-L includes three types of local neurons limited to the central ganglion. With ipsilateral dendritic arborizations and contralateral axonal branching, their gross morphology is similar to that of cricket omega cells. Interneuron PTG-DC, with the cell body in the prothoracic ganglion (PTG) and a contralaterally descending axon, conveys information received by the sensory organs of the front contralateral leg to the neuropil regions of the ipsilateral middle and hind legs. Based on their frequency tuning and acceleration sensitivity, the vibratory interneurons fall into two groups: the low-frequency units are tuned to 50 Hz and the middle frequency units to 200 Hz, with their acceleration thresholds at 10(-1) m/s(2) and 5 x 10(-3) m/s(2), respectively. Their function is discussed with relevance to the vibratory communication of N. viridula.

Vibrational communication along plants by the stink bugs Nezara viridula and Murgantia histrionica.

The velocity and spectral characteristics of vibrational signals of Nezara viridula (L.) and Murgantia histrionica (Hahn) (Heteroptera: Pentatomidae) were analyzed as the signals were transmitted through different plants. The velocity parameter of the body vibrations ranges from 0.1 to 1 mm/s. According to the mechanical properties of different substrates, the signal is attenuated or amplified during transmission from the insect's body to the substrate. Attenuation of up to 20 dB occurs during transmission of signals from leaves to stalks or stems. The velocity decrease with distance is below 0.5 dB/cm during transmission through less dense green stems, whereas it ranges between 0.6 and 1.6 dB/cm during transmission through more dense, woody stems. Signal velocity decreases non-linearly with increasing distance from the signal source. Regularly repeated velocity minima (nodes) and maxima (internodes) spaced 10-15 cm apart are characteristic of signal transmission through green plants but not woody stems. The signal velocity at some internodes exceeds the input value for N. viridula but not M. histrionica signals. The relative amplitude of the dominant frequency spectral peak varies with distance, along with overall signal velocity. Variable ratios of spectral peak amplitudes are characteristic for signals recorded at different distances from the source.

Tuning of host plants with vibratory songs of Nezara viridula L (Heteroptera: Pentatomidae).

Songs of the southern green stink bug Nezara viridula L, recorded on a non-resonant loudspeaker membrane, reflect frequency characteristics of body vibrations. The song dominant frequency directly depends on the repetition rate of potentials recorded from synchronously contracting muscles that vibrate the abdomen during singing. Spectra of naturally emitted signals recorded on the pronotum of a singing bug or on a plant contain peaks characteristic of plant resonant spectra. The dominant resonant frequency of sound-induced vibrations in bean and other stink bug host plants ranges between 160 and 215 Hz and subdominant peaks do not exceed 600 Hz. There is no correlation between spectral peak positions and recording points on a plant. The dominant resonance peak corresponds to the best frequency sensitivity of N. viridula middle frequency subgenual receptor cell. Subdominant peaks around and below 100 Hz lie close to the dominant frequency of body vibrations during singing and to the range of best frequency sensitivity of low frequency receptor cells. Tuning of plant resonant frequencies with spectral properties of songs and frequency sensitivity of sensory organs is discussed in the context of stink bug substrate-borne communication.