Behavioral response elicited by stimulation of the mesolimbic system after procaine and bicuculline injection into the pedunculopontine tegmental nucleus in rats.

The pedunculopontine tegmental nucleus (PPN) is anatomically connected with dopaminergic cells in the ventral tegmental area (VTA). In the present study, VTA-stimulation induced feeding or locomotor response was tested after temporary inactivation (procaine injection) or activation (bicuculline injection) of the PPN in the ipsi- or contralateral hemisphere. Motor and motivation aspects of appetitive behavior were analyzed on the basis of the latency/stimulation frequency curve shift paradigm, in male Wistar rats (n=48). Procaine injections into the PPN had more significant effects on both types of behavioral response during VTA stimulation than bicuculline. On the day of injection (day 0) procaine increased reaction threshold of the observed responses: a rise by 22% after contra- and 17% after ipsilateral injection in the case of feeding, and an inverse result side-wise for locomotor response, i.e. around 12% and 20% respectively. Bicuculline injected into the PPN did not cause significant effects on day 0 and the values of reaction threshold oscillated around ±10% for both behaviors, except in rats with locomotor reaction after contralateral injection. The observed reactions stabilized within on consecutive days (days 1-3) after procaine/bicuculline injection in both behavioral groups. The results indicate that the PPN and VTA belong to the same central circuitry involved in the regulation of psychomotor activation. However, the influence of PPN-VTA inter-hemispheric connections on reward and addiction function of the VTA is still unexplained.

Locomotor response to novelty correlates with the number of midbrain tyrosine hydroxylase positive cells in rats.

The present study investigated whether the higher dopaminergic system activation in rats with high (HRs) rather than low (LRs) locomotor activity in response to novelty depend on the number of cells containing the enzyme tyrosine hydroxylase (TH(+)) and/or differences in the morphology of these cells. One week after the novelty test, brains from male Wistar rats (HRs and LRs) were collected and stained for TH expression (immunohistochemistry) and for morphological analysis (immunofluorescent staining). The morphology and total number of TH(+) cells was analyzed for each A9 (substantia nigra) and A10 (ventral tegmental area) group of the midbrain dopaminergic cells. We found that HRs had a higher total number of TH(+) cells in the whole midbrain dopaminergic region (A9-A10) and in the A9 group only than LRs. In particular midbrain dopaminergic groups of neurons, HR/LR differences were regionally specific: HRs had a higher total number of TH(+) cells in the A9, and in the anterior part of the A10. In contrast, the LRs had a higher number of TH(+) cells in the parabrachial pigmented nucleus (A10) and in the posterior part of the A9. There were no significant differences in the morphology of the midbrain dopamine neurons between HRs and LRs. Moreover, there was a positive correlation between the total number of TH(+) neurons and the locomotor activity score in response to novelty in the whole A9-A10 region and in the particular A9 group only. The results obtained indicate that the higher behavioral activation in resting conditions correlates with the higher number rather than changes in the morphology of the midbrain dopaminergic TH(+) cells. It supports findings on the higher level of dopaminergic system activation in high responders to novelty that depends on the number of midbrain dopaminergic TH(+) neurons.

Differential effect of procaine injection into the rostral and caudal part of the nucleus pontis oralis on hippocampal theta rhythm in urethane-anesthetized rats.

The nucleus reticularis pontis oralis (RPO) is a reticular structure important for the regulation of paradoxical sleep (PS). However, the data concerning the relation between the RPO and the main tonic indicator of PS, hippocampal theta rhythm, are contradictory: although electrical or cholinergic stimulation of the RPO evoked well-synchronized theta activity, the electrolytic lesion of the structure had no effect on theta. In our experiment, the effect of procaine injections into different parts of the RPO on the electrical activity of the hippocampus, as well as on tail pinch-elicited hippocampal theta rhythm was assessed in urethanized rats. Power spectral analysis was performed using a Fast Fourier Transform routine in 1-Hz and 3-Hz bands between 0.6 and 12 Hz frequency. We have found that unilateral procaine inactivation of neurons in the caudal part of the RPO blocked the sensory-elicited theta rhythm. The same injection into the rostral RPO either had no effect or evoked long-lasting episodes of theta rhythm without sensory stimulation. These results suggest functional diversity of the parts of the RPO in mechanisms underlying production of hippocampal theta.

Hippocampal theta rhythm after serotonergic activation of the pedunculopontine tegmental nucleus in anesthetized rats.

The pedunculopontine tegmental nucleus (PPN), as a part of reticular formation activating system, is thought to be involved in the sleep/wake cycle regulation, and plays an important role in the generation and regulation of hippocampal rhythmical slow activity. The activity of PPN can be modulated by serotonergic system, mainly through multiple projections from raphe nuclei, which can influence PPN neurons through different classes of 5-HT receptors. In the present study, the effect of intra-PPN injection of two serotonin agonists: 8-OH-DPAT and 5-CT, on hippocampal formation EEG activity was examined in urethane-anesthetized rats. The study found that the microinjections induced prolonged spontaneous theta rhythm in both hippocampi with a short latency. The results obtained suggest that local inhibition of presumably cholinergic neurons in the PPN acts as a trigger for hippocampal theta activity.

Lesion and stimulation of the ventral tegmental area increases cholinergic activity in the rat brain.

Our previous study indicated that unilateral lesion of the ventral tegmental area (VTA) facilitates contralateral VTA stimulation-induced feeding or exploration. The present study was aimed to determine the possible role of the central cholinergic systems in this effect. Immunohistochemistry for choline acetyltransferase (ChAT) was used to measure the number of active cholinergic neurons in their major groups (Ch1-Ch6) and in striatal regions in rats subjected to unilateral electrocoagulation and contralateral VTA electrical stimulation (L/S group) in comparison to the unilaterally stimulated (S), unilaterally lesioned (L) and sham (Sh) groups. The study showed that unilateral VTA lesion increased (as compared to Sh group) the number of ChAT+ neurons in the Ch1-Ch3 and unilateral VTA stimulation increased the number in the Ch1 and the ventral pallidum only. The most sensitive to these changes in the mesolimbic system were cholinergic structures providing hippocampal afferentation. Surprisingly, there was no significant increase in the number of ChAT+ neurons in the L/S group. The obtained results did not confirm any evident influence of the cholinergic systems on the VTA lesion-induced facilitation of the behavioral response evoked by contralateral VTA stimulation.

Lesion of the ventral tegmental area amplifies stimulation-induced Fos expression in the rat brain.

Unilateral lesions of the ventral tegmental area (VTA), the key structure of the mesolimbic system, facilitate behavioral responses induced by electrical stimulation of the VTA in the contralateral hemisphere. In search of the neuronal mechanism behind this phenomenon, Fos expression was used to measure neuronal activation of the target mesolimbic structures in rats subjected to unilateral electrocoagulation and simultaneously to contralateral electrical stimulation of the VTA (L/S group). These were compared to the level of mesolimbic activation after unilateral electrocoagulation of the VTA (L group), unilateral electrical stimulation of the VTA (S group) and bilateral electrode implantation into the VTA in the sham (Sh) group. We found that unilateral stimulation of the VTA alone increased the density of Fos containing neurons in the ipsilateral mesolimbic target structures: nucleus accumbens, lateral septum and amygdala in comparison with the sham group. However, unilateral lesion of the VTA was devoid of effect in non-stimulated (L) rats and it significantly amplified the stimulation-induced Fos-immunoreactivity (L/S vs S group). Stimulation of the VTA performed after contralateral lesion (L/S) evoked strong bilateral induction of Fos expression in the mesolimbic structures involved in motivation and reward (nucleus accumbens and lateral septum) and the processing of the reinforcing properties of olfactory stimuli (anterior cortical amygdaloid nucleus) in parallel with facilitation of behavioral function measured as shortened latency of eating or exploration. Our data suggest that VTA lesion sensitizes mesolimbic system to stimuli by suppressing an inhibitory influence of brain areas afferenting the VTA.

Modulation of hippocampal theta rhythm by the opioid system of the pedunculopontine tegmental nucleus.

The pedunculopontine tegmental nucleus (PPN) belongs to the brainstem system which synchronizes hippocampal activity. Theta relevant intra-PPN circuitry involves its cholinergic, GABA-ergic and glutamatergic neurons and Substance P as neuromodulator. Evidence that PPN opioid elements also modulate the hippocampal theta is provided here. In urethane-anesthetized rats a unilateral microinjection of morphine (MF) (1.5 and 5 microg) increased the maximal peak power of tail pinch-induced theta. The higher dose also increased the corresponding frequency. When the theta was evoked by intra-PPN injection of carbachol (10 microg), the addition of MF (5 microg) prolonged theta latency and shortened the duration of the theta. These effects of MF were blocked by naloxone (5 microg). The results obtained suggest that the PPN opioid system can enhance or suppress the hippocampal theta depending on the actual level of PPN activation.

Microinjection of procaine and electrolytic lesion in the ventral tegmental area suppresses hippocampal theta rhythm in urethane-anesthetized rats.

The midbrain ventral tegmental area (VTA), a key structure of the mesocorticolimbic system is anatomically connected with the hippocampal formation. In addition mesocortical dopamine was found to influence hippocampus-related memory and hippocampal synaptic plasticity, both being linked to the theta rhythm. Therefore, the aim of the present study was to evaluate the possible role of the VTA in the regulation of the hippocampal theta activity. The study was performed on urethane-anesthetized male Wistar rats in which theta rhythm was evoked by tail pinch. It was found that unilateral, temporal inactivation of the VTA by means of direct procaine injection resulted in bilateral suppression of the hippocampal theta which manifested as a loss of synchronization of hippocampal EEG and respective reduction of the power and also the frequency of the 3-6 Hz theta band. Depression of the power of the 3-6 Hz component of the EEG signal was also seen in spontaneous hippocampal EEG after procaine. The permanent destruction of the VTA by means of unilateral electrocoagulation evoked a long-lasting, mainly ipsilateral depression of the power of the theta with some influence on its frequency. Simultaneously, there was a substantial increase of the power in higher frequency bands indicating decrease of a synchrony of the hippocampal EEG activity. On the basis of these results indicating impairment of synchronization of the hippocampal activity the VTA may be considered as another part of the brainstem theta synchroning system.

Activation of tachykinin system in the pedunculopontine tegmental nucleus suppresses hippocampal theta rhythm in urethane-anesthetized rats.

The pedunculopontine tegmental nucleus (PPN) is one of the reticular generators of the hippocampal theta rhythm. The PPN neuronal circuitry related to theta generation involves its cholinergic, GABA-ergic and glutamatergic components. Here we provide data indicating that the PPN tachykinin system may also be a part of this circuitry. In the experimental model of the tail-pinch elicited hippocampal theta in urethane-anesthetized rats (implanted with bilateral recording electrodes in the stratum moleculare of the upper blade of the dentate gyrus and with injection cannula unilaterally inserted into the PPN) it was found that intra-PPN microinjection of Substance P (SP) and [d-Pro2, d-Phe7, d-Trp9]-Substance P (DPDPDT) caused suppression of the theta and enhancement of the delta activity in the hippocampal EEG. Accordingly, there was approximately a 50% (SP)-70% (DPDPDT) decline of the peak power in the theta frequency range and a decrease by 0.4 Hz in the corresponding peak frequency (DPDPDT only) in both hippocampi. The circuitry through which SP exerts its effect in the PPN can be only hypothetical at present. We suggest SP-evoked activation (either direct or indirect through the glutamatergic inputs) of the GABA interneurons which may tonically inhibit PPN outputs to the other theta-relevant structures.

Suppression of natural killer cell cytotoxicity following chronic electrical stimulation of the ventromedial hypothalamic nucleus in rats.

In our previous study we found that chronic electrical stimulation of the lateral hypothalamus (LH) enhances and its lesion suppresses natural killer cell cytotoxicity (NKCC) and a large granular lymphocyte (LGL) number in conscious, freely behaving rats. Since the ventromedial nucleus of the hypothalamus (VMH) is regarded as behaviorally and physiologically opposite to LH, in our present study we investigated whether this antagonism also holds for the immune functions. Chronic electrical VMH stimulation effect on 1) immune parameters: both spleen and blood NKCC (chromium release assay and single-cell agarose assay) and the number of large granular lymphocytes (LGL; a morphological method), and 2) endocrine parameters: immunosuppressive-corticosterone (COR) and testosterone (TST) and immunostimulative-growth hormone (GH) and prolactin (PRL) plasma levels (RIA) was assessed. Twenty-one days of electrical stimulation of VMH caused significant decrease in both spleen and blood NKCC at the population level (chromium release assay) but not at the single cell level (agarose assay) with a simultaneous fall in the LGL number. Rats responding to the VMH stimulation with behavioral inactivation (BIN) showed a significantly lower depression of NKCC and LGL number than those responding with an aversive reaction (AVE). Depression of NKCC coexisted with various hormonal changes: increase of PRL, increase (AVE) or fall (BIN) of COR, decrease of GH (BIN), and increase of TST (VMH-stimulated and VMH-sham). There were significant differences in all measured plasma hormones between BIN and AVE groups. The results obtained indicate that VMH decreases cell-mediated immune response, represented by NK cell activity. The immunosuppressive effect is dependent on the behavioral outcome of VMH stimulation (BIN/AVE) rather than tested endocrine variables. Moreover, the present results indicate that the VMH and LH are antagonistically engaged in the regulation of NK cell cytotoxicity.

Chronic electric stimulation of the midbrain ventral tegmental area increases spleen but not blood natural killer cell cytotoxicity in rats.

Previously we found that in conscious, freely behaving rats chronic electric stimulation of the lateral hypothalamus (LH) caused significant augmentation of natural killer cell cytotoxicity (NKCC) and a large granular lymphocyte (LGL) number more pronounced in the spleen than in the peripheral blood. The LH belongs to the so-called "brain reward system", a collection of the central structures whose activation produce positive emotions. The midbrain ventral tegmental area (VTA) is another prominent reward-relevant structure. In the present work, chronic electric stimulation of VTA (constant current 0.1 ms duration cathodal pulses delivered at frequency 50 Hz during 60 min daily session for 14 consecutive days) caused in rats an increase in the spleen but not in the peripheral blood NKCC (chromium release assay) without simultaneous effect on the number of large granular lymphocytes (LGL) (morphological method) and plasma level of prolactin (PRL), growth hormone (GH), corticosterone (COR), and testosterone (TST). This effect was anatomically specific as no influence of analogous thalamic stimulation on immune and endocrine response was found. The results obtained indicate that both reward-related areas VTA and LH enhance the cell-mediated immune response, represented by natural killer cytotoxicity, especially in the spleen. However, the effect pronounced by VTA is weaker than that of LH, possibly due to additional connections of LH with the hormonal and/or autonomic control systems.

The effects of lateral hypothalamic lesions on peripheral blood natural killer cell cytotoxicity in rats hyper- and hyporesponsive to novelty.

Individual variability in the central control of the cellular immune responses is the main subject of the study. Previously, it was found that destruction of the lateral hypothalamus (LH) produced long-term depression of the cytotoxicity of NK cells (NKCC) and their number (LGL). In the present experiment we compared changes in the peripheral blood NKCC, LGL number, as well as leukocyte and lymphocyte number, their mitogenic activity and plasma corticosterone level evoked by electrolytic LH lesions in rats which were categorized as either high (HR) and low (LR) responders according to their locomotor response to a new environment. It was found that: (1) before the lesion NKCC (measured by 51Cr release assay) was higher in the HRs than in LRs; (2) LH damage caused a drop in NKCC and LGL number (21st postlesion day) preceded by a transient enhancement (5th postlesion day) significant for HRs only. As a result of a greater decrease in the HRs than LRs the baseline differences between groups disappeared by 21st postlesion day; (3) NKCC and LGL depression was not accompanied by changes in lytic activity of a single NK cell (agarose assay) which indicates that NKCC decrease concerned the population level and was dependent on LGL redistribution and/or recycling rate; (4) on the 21st postlesion day there was a significant leuko- and lymphopenia in the lesioned groups both HRs and LRs; (5) proliferative lymphocyte response to PWM (colorimetric assay) and plasma corticosterone level were not affected either by the motility level or by the lesion. The results emphasize the importance of individual differences in behavioral reactivity for NKCC regulation and a possible involvement of LH in the mechanism which connects high locomotor activity with stimulation of NKCC.

Chronic electrical stimulation of the lateral hypothalamus increases natural killer cell cytotoxicity in rats.

Previously, we found that in rats coagulation of the lateral hypothalamus (LH) caused depression of the peripheral blood natural killer cell cytotoxicity (NKCC) and the number of large granular lymphocytes (LGL). In the present work, we have tested the effects on both spleen and blood NKCC of acute (1 day) and chronic (21 days) electrical stimulation of LH, and LGL number in conscious, freely behaving animals. Five groups of male Wistar rats were used: LH stimulated (n=22), thalamic (Thal) stimulated control (n=4), operated but non-stimulated LH sham controls (n=7), non-operated normal control group (n=8) and spleen baseline group (n=10). Chronic stimulation of LH caused significant augmentation of NKCC (51Cr-release assay) and LGL number (a morphological method), more pronounced in the spleen than in the peripheral blood. Rats responding to LH stimulation with feeding showed a slightly greater effect than those responding with a locomotor reaction. The observed effects were anatomically specific as no influence of Thal stimulation or the sham procedure was found. The results are discussed in terms of the involvement of LH in reward phenomena and the hormonal control of the immune system.

Effect of unilateral ibotenate lesions of the ventral tegmental area on cortical and hippocampal EEG in freely behaving rats.

It was found previously that unilateral destruction of the ventral tegmental area (VTA) facilitated behavioral responses (exploration, eating) induced by electrical stimulation of the contralateral VTA. The same effect occurred after unilateral injections of pharmacological agents, which led to a decrease in dopaminergic transmission in the VTA. While trying to explain the mechanism behind this "contralateral facilitation effect" in the present experiment we examined whether augmentation of function of the contralateral hemisphere would be reflected in cortical and hippocampal EEG changes in conscious rats. Unilateral, cytotoxic lesion of the VTA caused a bilateral decrease in neocortical and hippocampal EEG power during both exploratory sniffing and eating. Depression involved all the frequency bands in the prefrontal cortex, mainly in the hemisphere contralateral to the VTA lesion. In the hippocampus the depression was slightly more intense ipsilaterally, also involving all the frequency bands although to different degrees. The results indicate that the VTA is involved in the regulation of cortical and hippocampal activity during VTA-dependent behavioral activation, and that the "contralateral facilitation effect" is concomitant with lateralized changes in EEG activity.