State-dependent effects of some neuropeptides and neurotransmitters on neuronal activity of the medial septal area in brain slices of the ground squirrel, Citellus undulatus

Neuronal activity of the medial septal area was recorded extracellularly in brain slices taken from hibernating (winter) and waking (summer) ground squirrels. The effects of neuropeptides identified in the brain tissue of hibernators (Thr-Ser-Lys-Tyr, Thr-Ser-Lys-Tyr-Arg and Asp-Tyr) on the background activity and responses to electrical stimulation of the median forebrain bundle were analysed. For comparison, the effects of bath application of noradrenaline and serotonin were also tested. Spontaneous activity in half of all neurons (47-56%) was changed under the influence of neuropeptides in hibernating ground squirrels, while in waking ground squirrels the proportion of responsive neurons was significantly lower (25-30%). The tendency for higher efficacy in hibernating ground squirrels was observed for serotonin; only noradrenaline was equally effective in both groups of animals. Electrically evoked responses of the medial septal nucleus-nucleus of the diagonal band neurons were also strongly modulated by neuropeptides; their changes could occur in the absence of shifts in the level and pattern of spontaneous activity. All three neuropeptides had differential action on the level of spontaneous activity, as well as on inhibitory and excitatory components of electrically evoked responses. Thus, the character and distribution of the effects were state dependent and differed greatly in hibernating and waking ground squirrels. The experiments confirmed that medial septal nucleus-nucleus of the diagonal band neurons have higher excitability and responsiveness to some neuropeptides and neurotransmitters in hibernating ground squirrels.The data obtained suggest an increased latent excitability and responsiveness of septal neurons during hibernation and their possible active participation in urgent arousal under the influence of sensory signals.     

Seasonal changes in the intestinal immune system of hibernating ground squirrels

Hibernation is associated with a prolonged fast (5-8 mo) which has the potential to affect intestinal immunity. We examined several aspects of the intestinal immune system in summer (non-hibernating) and hibernating ground squirrels. Peyer's patches were largely unaffected by hibernation, but numbers of intraepithelial lymphocytes (IEL) and lamina propria leukocytes (LPL) were greater in hibernators compared with summer. Hibernator IEL were less mature as demonstrated by low numbers of cells expressing activation-associated markers and co-receptors. Compared with summer, the percentage of B cells was higher and percentage of T cells was lower in the hibernator LPL. Hibernation was associated with greater mucosal levels of IFN-gamma, TNF-alpha, IL-10 and IL-4, but IL-6 and TGF-beta were unchanged. Mucosal IgA levels were greater in entrance and torpid hibernators compared with summer. The results suggest that modifications of the intestinal immune system during hibernation may help preserve gut integrity throughout the winter fast.     

State-dependent activity of neurons in the perifornical hypothalamic area during sleep and waking

Neurons containing orexins are located in the perifornical hypothalamic area and are considered to have a role in sleep-wake regulation. To examine how this area is involved in the regulation of sleep and wakefulness, we recorded neuronal activity in undrugged, head-restrained rats across sleep-waking cycles. Recordings were made in the perifornical hypothalamic area where orexin-immunoreactive neurons are distributed (PFH), and in the area dorsal to the PFH, including the zona incerta and subincertal nucleus (collectively referred to as ZI). The 40 neurons recorded from in the PFH were divided into five groups: (1) neurons most active during paradoxical sleep (PS, n=14, 35%), (2) neurons active during both waking (W) and PS (n=12, 30%), (3) neurons most active during W (n=7, 18%), (4) neurons most active during slow-wave sleep (SWS, n=3, 7.5%), and (5) neurons whose activity had no correlation with sleep-waking states (n=4, 10%). Of 30 neurons recorded from in the ZI, the corresponding numbers were 13 (43%), seven (23%), six (20%), three (10%), and one (3.3%). In both areas, neuronal activity fluctuated more during PS than during W. Waking-specific neurons (group 3) in the PFH generated action potentials with longer durations than those produced by other types of neurons. About half of the neurons in the PFH that were classified in groups 1, 2, and 3 increased their firing rate after the transition from one state to another, while higher percentages of neurons of groups 1 and 2 in the ZI than those in the PFH increased their firing rate prior to the state shift from SWS to PS. In these ZI neurons, however, the firing rate varied considerably at the state shift.These results suggest that the PFH and ZI are involved in the regulation of PS or W, especially the regulation of phasic events during PS or the maintenance of W. The ZI appears to be more closely involved than the PFH in the induction of PS or some phasic phenomena associated with PS.     

Systemic morphine and local opioid effects on neuronal activity in the medial prefrontal cortex

Behavioral studies support the importance of the medial prefrontal cortex in the circuitry of drug-reinforced behavior, yet the neurophysiological correlates of this phenomenon remain unclear. The present study evaluates opioid neuropharmacology in the medial prefrontal cortex of the anesthetized rat. The effects of both systemic and local application of mu agonists on individual neurons in the medial prefrontal cortex were examined. Systemic morphine was found to inhibit (63%), excite (4%) or have no effect on (33%) spontaneous firing. The inhibitory response was reversed by systemic naloxone in 77% of the cases. Electrophoretic application of a mu-selective agonist, ]d-Ala², N-Me-Phe⁴,Gly-ol⁵]enkephalin, had mixed effects on cell activity. While most cells exhibited no change in firing rate (53%), 38% showed inhibition of spontaneous activity. The ]d-Ala²,N-Me-Phe⁴,Gly-ol⁵]enkephalin-evoked inhibitory responses were antagonized by electrophoresis of naloxone (86%).

These results indicate that the medial prefrontal cortex might directly mediate some portion of the overall response to opiates in reinforcement or self-administration paradigms. The naloxone-reversible inhibition of firing seen following both systemic and local application of predominantly mu-selective agonists argues for a direct involvement of medial prefrontal cortical neurons in opiate-induced effects. However, the smaller percentage of cells inhibited by local versus systemic application of mu agonists also supports an influence of other brain circuitry in this response.     

Paradoxical state-dependent excitability of the medial septal neurons in brain slices of ground squirrel, Citellus undulatus

Spontaneous and evoked neuronal activity of the medial septum-diagonal band complex was investigated extracellularly in slices, taken from the brain of the three groups of animals: hibernating ground squirrels, waking ground squirrels, and guinea-pigs. All slices were incubated at 31-32 degrees C. The slices of the ground squirrels' brain were retested after keeping them for 15-36 h in the refrigerator at 2-4 degrees C. In all experimental groups the majority of the medial septum-diagonal band complex neurons had high regular or rhythmic burst spontaneous activity, which in half of the neuronal population persisted in conditions of synaptic blockade. The low-frequency irregular activity of the surrounding structures (lateral septum, caudate, accumbens, medial preoptic area) was completely suppressed in these conditions. The density of the spontaneously active neurons in the slices, as well as the mean frequency of discharges in the medial septum-diagonal band complex of hibernating ground squirrels, was significantly higher than that in waking ground squirrels and guinea-pigs. Stimulation of the medial forebrain bundle evoked initial suppression of activity in majority of the medial septum-diagonal band complex units; in many of them the suppression was followed by a burst discharge. The neurons with background rhythmic burst activity always responded by resetting the spontaneous bursts. In total, about 50-60% of the medial septum-diagonal band complex neurons of waking ground squirrels and guinea-pigs responded by post inhibitory bursts to the stimulation of medial forebrain bundle, while in hibernating ground squirrels such responses were observed in nearly all neurons. The threshold values of the stimulating current were significantly lower in the hibernating ground squirrels' group, the mean duration of the initial suppression was shorter, the intraburst density of spikes and/or duration of the bursts was increased. Thus, evaluation of spontaneous and evoked activity on the basis of various criteria revealed surprising similarity between the two groups of active animals, while the activity and excitability of the medial septum-diagonal band complex neurons was approximately doubled in the hibernating animals. This difference between active and hibernating ground squirrels was preserved during retesting after deep and prolonged cooling of the slices. The experiments demonstrate paradoxical stable increase of activity and excitability of the medial septum-diagonal band complex neurons in the hibernating ground squirrels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Noise-induced changes of neuronal spontaneous activity in mice inferior colliculus brain slices

The inferior colliculus (IC) in vivo is reportedly subject to a noise-induced decrease of GABA-related inhibitory synaptic transmission accompanied by an amplitude increase of auditory evoked responses, a widening of tuning curves and a higher neuronal discharge rate at suprathreshold levels. However, other in vivo experiments which demonstrated constant neuronal auditory thresholds or unchanged spontaneous activity in the IC after noise exposure did not confirm those findings. Perhaps this can be the result of complex noise-induced interactions between different central auditory structures. It was, therefore, the aim of the present study to investigate the effects of noise exposure on the spontaneous electrical activity of single neurons in a slice preparation of the isolated mouse IC. Normal hearing mice were exposed to noise (10 kHz center frequency at 115 dB SPL for 3 h) at the age of 21 days under anesthesia (Ketamin/Rompun 10:1). After one week, auditory brainstem response (ABR) recordings and extracellular single-unit recordings from spontaneously active neurons within the IC slice were performed in noise-exposed and in normal hearing control mice. Noise-exposed animals showed a significant ABR threshold shift in the whole tested frequency range and a significant lower neuronal spontaneous activity in all investigated isofrequency laminae compared to controls. In both groups, the firing rate of 80% of IC neurons (approximately) increased significantly during the application of the GABA(A) receptor antagonist Bicucullin (10 microM). The present findings demonstrate a noise-related modulation of spontaneous activity in the IC, which possibly contribute to the generation of noise-induced tinnitus and hearing loss.     

Sleep during arousal episodes as a function of prior torpor duration in hibernating European ground squirrels

EEG's were recorded in hibernating European ground squirrels during euthermic arousal episodes at an ambient temperature of 5.5°C. Spontaneous torpor bouts ranged from 6 to 15 days, body temperature during torpor was 7.5°C. The torpor duration prior to EEG measurements was experimentally manipulated: the animals were induced to arouse by gentle handling after torpor of less then 1 day (n=3), 1–2 days (n=6), 3–4 days (n=9) and 5–12 days (n=9). The animals slept 71.5% of euthermic time, of which 61.4% NREM and 10.2% REM sleep. NREM percentage was slightly positively and REM percentage negatively correlated with prior torpor duration (TD). Spectral analysis showed changes in EEG activity during the euthermic phase in the slow wave frequency range (1–4 Hz) and in higher frequencies. Prior TD specifically affected the slow waves. Slow wave activity decreased exponentially during the euthermic phase. The initial slow wave activity showed a systematic increase with prior TD, which could be described by an exponentially saturating function, albeit with a relatively small time constant compared with spontaneous torpor duration. It is concluded that sleep during arousal episodes following torpor at an ambient temperature of 5.5°C is affected both in structure and intensity by prior TD. The results are consistent with the proposition that torpor inhibits the restorative function of sleep.     

Intrathecal methiothepin and thyrotropin-releasing hormone effects on penile erection

Intrathecal thyrotropin-releasing hormone (TRH) potently inhibits penile erection at all doses (100, 500, 1000 or 5000 pmol) tested so far. Since the serotonin receptor antagonist methiothepin (MT) inhibits TRH responses in other systems, this study tested the hypothesis that MT-sensitive receptors mediate the effect of TRH on penile erection in rats. When compared to controls, the highest doses of IT TRH (0, 10 or 500 pmol) or MT (5 or 50 nmol) significantly altered penile reflex latency. When coadministered (50 nmol MT/500 pmol TRH), the effect of TRH was reversed, suggesting that the high dose of MT antagonized the inhibitory actions of TRH. The low dose of MT (5 nmol) did not block the 500 pmol TRH inhibition of reflex latency. These data further suggest that MT sensitive receptors are important in (1) mediating normal penile reflexes and (2) mediating the inhibitory response to TRH.     

Modulatory actions of luteinizing hormone-releasing hormone on electrical activity of preoptic neurons in brain slices

Single unit activity was recorded from 378 neurons, in two preoptic nuclei rich in luteinizing hormone-releasing hormone neurons, using in vitro brain tissue slices which were prepared form either ovariectomized or ovariectomized plus estradiol-treated rats. To test possible transmitter-like actions, agents were injected into the perfusion medium. Luteinizing hormone-releasing hormone excited 46%, inhibited 7%, and evoked biphasic responses in 2% of the 250 units tested. By comparison, two other peptides, thyrotropin-releasing hormone and cholecystokinin-octapeptide sulfated were exclusively excitatory, acting on 55 and 67% of the neurons, respectively. The response to thyrotropin-releasing hormone, cholecystokinin-octapeptide sulfated, and neurotransmitters were prompt, large, and consistent from trial to trial. In contrast, responses to luteinizing hormone-releasing hormone were usually delayed, small, and variable. Responses to the agents tested were not affected by in vivo estradiol treatment. Possible modulatory actions of luteinizing hormone-releasing hormone were tested by comparing the responses of single neurons to norepinephrine and serotonin before and after an application of luteinizing hormone-releasing hormone. In 39 and 20% of the 119 neurons tested, the norepinephrine responses were potentiated and attenuated, respectively, by luteinizing hormone-releasing hormone. In 46 serotonin-responsive neurons, 28% were potentiated and 22% attenuated. These neuromodulatory actions of luteinizing hormone-releasing hormone were specific in affecting only certain responses of certain neurons, and they were not duplicated on the same neurons by thyrotropin-releasing hormone. It appears that luteinizing hormone-releasing hormone may be a neuromodulator in the preoptic area.     

Functional stability of the brain slices of ground squirrels, Citellus undulatus, kept in conditions of prolonged deep periodic hypothermia: electrophysiological criteria

The brain of hibernating animals, controlling the physiological functions during the hibernation cycles, is itself subject to deep cooling during bouts of hibernation. This suggests its high tolerance to deep hypothermia. Effects of prolonged deep cooling were investigated in hippocampal and septal slices, taken from the brains of three groups of animals: hibernating ground squirrels, actively waking ground squirrels, and guinea-pigs. The slices were kept at a low temperature (2-4 degrees C) for various periods of time (from several hours up to six days) and periodically tested in warm (31 degrees C) incubation medium. The hippocampal field potentials (mainly of field CA1), as well as spontaneous activity of single neurons of hippocampus and medial septum were recorded. For comparative purposes mean amplitudes of population spikes and mean frequency of spontaneous neuronal discharge were used. Significant differences between the experimental groups were observed in recovery of functional activity of the slices after their dissection from the brain, as well as after deep cooling. In both cases re-establishment of neuronal activity in ground squirrels occurred more rapidly, than in guinea-pigs. The most dramatic was the difference in maximal time of survival of the slices under conditions of deep cooling. Independently of periodicity of the electrophysiological testing in warm medium, the slices taken from hibernating squirrels retained their activity for seven to nine days, the slices of waking ground squirrel hippocampus survived up to six to seven days, while those of guinea-pis did not recover their functional activity after cooling for more than one to two days.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of pentylenetetrazole-induced kindling on thyrotropin-releasing hormone biosynthesis and receptors in rat brain

It has been postulated that changes in thyrotropin-releasing hormone biosynthesis may be involved in the mechanism of kindling--an animal model of epileptogenesis. To test this hypothesis, a time-course study was carried out to investigate the effects of pentylenetetrazole kindling (40 mg/kg i.p., daily for eight days) on the expression of gene coding for preprothyrotropin-releasing hormone, the thyrotropin-releasing hormone tissue level and thyrotropin-releasing hormone receptor parameters in rat brain. As shown by an in situ hybridization study, a single, convulsant dose of pentylenetetrazole (70 mg/kg i.p.) increased the preprothyrotropin-releasing hormone messenger RNA level in the dentate gyrus of the hippocampal formation and piriform cortex after 3 h and, to a greater extent, after 24 h. Those changes were accompanied with increases in the thyrotropin-releasing hormone level in the striatum, hippocampus, amygdala and piriform cortex. Seven days after single pentylenetetrazole administration, the thyrotropin-releasing hormone level was still significantly elevated in the piriform cortex and striatum. Acute pentylenetetrazole decreased the density (Bmax) of thyrotropin-releasing hormone receptors in the striatum after 3 and 24 h, and increased that density in the piriform cortex and amygdala after 24 h and seven days, respectively. The thyrotropin-releasing hormone receptor affinity (Kd) was decreased in the striatum and increased in the amygdala after only 3 h. Kindled rats showed a moderate increase in the preprothyrotropin-releasing hormone messenger RNA content in the dentate gyrus of the hippocampal formation and piriform cortex after 3 and 24 h; however, a significant decrease in those parameters was found after 14 days. After 3 and 24 h, pentylenetetrazole kindling also elevated the thyrotropin-releasing hormone content in the hippocampus, piriform cortex, and striatum (in the latter structure after 24 h only), whereas in the septum the thyrotropin-releasing hormone level was decreased. After seven days, the thyrotropin-releasing hormone level was still elevated in the hippocampus and piriform cortex of kindled rats, but after 14 days it was significantly lowered in the hippocampus. The kindled rats also showed a significant decrease in the density (Bmax) of thyrotropin-releasing hormone receptors in the striatum (after 24 h, seven and 14 days), and an increase in the piriform cortex (after seven days). The thyrotropin-releasing hormone receptor affinity (Kd) value was increased in the hippocampus after seven and 14 days, and in the piriform cortex after seven days. These results indicate that pentylenetetrazole kindling induces long-lasting alterations in the thyrotropin-releasing hormone biosynthesis and thyrotropin-releasing hormone receptor affinity in discrete regions of rat brain. These region-specific changes, in particular down-regulation of the thyrotropin-releasing hormone biosynthesis in the hippocampus, may be involved in chronic neuronal hyperexcitability associated with kindling.     

Erratum to "Noise-induced changes of neuronal spontaneous activity in mice inferior colliculus brain slices"

The inferior colliculus (IC) in vivo is reportedly subject to a noise-induced decrease of GABA-related inhibitory synaptic transmission accompanied by an amplitude increase of auditory evoked responses, a widening of tuning curves and a higher neuronal discharge rate at suprathreshold levels. However, other in vivo experiments which demonstrated constant neuronal auditory thresholds or unchanged spontaneous activity in the IC after noise exposure did not confirm those findings. Perhaps this can be the result of complex noise-induced interactions between different central auditory structures. It was, therefore, the aim of the present study to investigate the effects of noise exposure on the spontaneous electrical activity of single neurons in a slice preparation of the isolated mouse IC. Normal hearing mice were exposed to noise (10 kHz center frequency at 115 dB SPL for 3 h) at the age of 21 days under anesthesia (Ketamin/Rompun 10:1). After one week, auditory brainstem response (ABR) recordings and extracellular single-unit recordings from spontaneously active neurons within the IC slice were performed in noise-exposed and in normal hearing control mice. Noise-exposed animals showed a significant ABR threshold shift in the whole tested frequency range and a significant lower neuronal spontaneous activity in all investigated isofrequency laminae compared to controls. In both groups, the firing rate of 80% of IC neurons (approximately) increased significantly during the application of the GABA(A) receptor antagonist Bicucullin (10 microM). The present findings demonstrate a noise-related modulation of spontaneous activity in the IC, which possibly contribute to the generation of noise-induced tinnitus and hearing loss.     

Differential effects of protein kinase C on neuronal nitric oxide synthase activity in rat cerebellar slices and in vitro

The effects of neuronal nitric oxide synthase (NOS) of protein kinase C (PKC) activation in rat cerebellar slices and of in vitro phosphorylation by PKC were compared. Incubation of slices with 1-aminocyclopentane-1,3-trans-dicarboxylic acid (trans-ACPD) or phorbol myristate acetate (PMA) in the presence of okadaic acid (OA) shifted the calcium sensitivity of neuronal NOS in the homogenate or in the cytosolic fraction. trans-ACPD promoted translocation of PKC activity to the particulate fraction in the slices. PMA in the presence of OA enhanced phosphorylation of GAP43 protein in the slices. These results ensured that both treatments activated PKC in the slice. However, when neuronal NOS in the slice treated with PMA and OA, in which GAP43 phosphorylation was detected, was immunoprecipitated by a specific antibody, no indication of neuronal NOS phosphorylation was obtained. Nevertheless, PKC phosphorylated partially purified neuronal NOS in vitro. Phosphorylated neuronal NOS showed greater activity than unphosphorylated NOS, but their calcium sensitivity was identical. These data indicated that neuronal NOS is not susceptible to PKC-dependent phosphorylation in cerebellar slices and that the calcium-sensitivity shift of neuronal NOS takes place without direct phosphorylation of neuronal NOS, suggesting the involvement of unknown proteins whose phosphorylation would regulate the calcium sensitivity of neuronal NOS in the cerebellum.