Sham-stimulated Rats Research Articles

Gastroprotective effect of electrical spinal cord stimulation may be mediated by glucocorticoids produced in response to this stimulation

We demonstrated previously that stress-induced glucocorticoids are gastroprotective hormones but not ulcerogenic ones (Filaretova et al., 2016). Recently electrical spinal cord stimulation began to be used for both experimental studies of motor functions regulation and rehabilitation of motor functions in patients with spinal cord injury (Gerasimenko et al., 2021; Moshonkina et al., 2016). The spinal cord stimulation directed to the activation of spinal locomotor related networks affected visceral systems as well. Recently in our teamwork we have shown that the electrical spinal cord stimulation can cause an increase in blood glucocorticoid level accompanied by a gastroprotective effect (Filaretova et al., 2022). The aim of the present work was to verify the hypothesis that the gastroprotective effect of the electrical spinal cord stimulation may be mediated by glucocorticoids produced in response to this stimulation. Gastric injury was induced in anesthetized rats by prolonged gastric ischemia/reperfusion (I/R, 30 min occlusion of celiac artery followed by 3 h of reperfusion) or indomethacin administration (IM, 35 mg/kg, sc). For spinal stimulation, stainless steel wire electrodes were fixed in fasciae between T11-12 and L1–L2 vertebrae of anesthetized animals. Motor evoked responses in hindlimbs muscles to spinal stimulation were recorded. Spinal stimulation at 30 Hz was carried out with a subthreshold current for inducing muscle contractions (80% of the threshold). Sham-stimulated (control) rats were subjected to the same manipulations except for the stimulation itself. To test the participation of glucocorticoids in the gastroprotective effect of the stimulation, two approaches were used: pretreatment by the inhibitor of glucocorticoid synthesis, metyrapone (30 mg/kg, i.p.) and the antagonist of glucocorticoid receptors RU-38486 (20 mg/kg, i.p.). The concentration of corticosterone in blood plasma was determined using commercial ELISA kits. In control rats the spinal cord stimulation resulted in an increase of blood corticosterone level 1 h later. The spinal stimulation, applied 1 h before the onset of ulcerogenic stimuli, also significantly attenuated gastric erosion formation induced by I/R and IM as well. Metyrapone injected shortly before the stimulation caused a fast inhibition of corticosterone response and reversed the gastroprotective effect stimulation. The gastroprotective effect of the stimulation was also prevented by the pretreatment rats with glucocorticoid receptor antagonist RU-38486. The obtained results indicate on contribution of glucocorticoids to gastroprotective effect of spinal cord stimulation. In conclusion, the findings support the hypothesis that the gastroprotective effect of the electrical spinal cord stimulation may be mediated by glucocorticoids produced in response to this stimulation. The study was supported by the Ministry of Education and Science of the Russian Federation (agreement No. 075-15-2020-921 for the creation and development of the world-class scientific center “Pavlov Center “Integrative Physiology - to medicine, high-tech healthcare and technologies of stress resistance”). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effects of Electrical Spinal Cord Stimulation on the Activity of the Hypothalamic‐Pituitary‐Adrenocortical System and the Sensitivity of the Gastric Mucosa to Ulcerogenic Stimuli

Our previous results of two areas of unrelated researches are the basis of the present study. We demonstrated previously that the hypothalamic‐pituitary‐adrenocortical (HPA) axis is gastroprotective component of the brain‐gut axis and stress‐induced glucocorticoids are gastroprotective hormones but not ulcerogenic ones (Filaretova et al., 2016). At the same time recently electrical spinal cord stimulation began to be used for both experimental studies of motor functions regulation and rehabilitation of motor functions in patients with spinal cord injury (Gerasimenko et al., 2021; Moshonkina et al., 2016). The spinal cord stimulation directed to the activation of spinal locomotor related networks affected visceral systems as well. The aim of the present experimental work was to verify whether electrical spinal cord stimulation may activate the HPA axis and affect a sensitivity of the gastric mucosa to ulcerogenic stimuli in rats. Two ulcerogenic stimuli were used in anesthetized rats: 1) prolonged gastric ischemia/reperfusion (I/R, 30 min occlusion of celiac artery followed by 3 h of reperfusion); 2) indomethacin administration (IM, 35 mg/kg, sc). For spinal stimulation, stainless steel wire electrodes (AS632; Cooner Wire, California, USA) were fixed in fasciae between T11‐12 and L1–L2 vertebrae of anesthetized animals. Electrodes for recording motor responses to stimulation placed to the muscles of the hind limbs. A‐M System, Model 2100 stimulator was used for spinal cord stimulation. At the beginning motor thresholds determined by stimulation with monopolar rectangular pulses (1 ms). Then the spinal cord was stimulated with a frequency 30 Hz and a subthreshold current (350–700 μA) in order to exclude the influence of movements for 20 min. Sham‐stimulated (control) rats were subjected to the same manipulations except for the stimulation itself. The concentration of corticosterone in blood plasma was determined using commercial ELISA kits. The spinal cord stimulation resulted in an increase of blood glucocorticoid level 1 h later suggesting an activation of the HPA axis. The spinal stimulation, applied 1 h before the onset of ulcerogenic stimuli, also significantly attenuated gastric erosion formation induced by I/R and IM as well. The data suggest that the electrical spinal cord stimulation may activate the HPA axis and affect a sensitivity of the gastric mucosa to ulcerogenic stimuli in rats producing gastroprotective effect. We consider the results as a basis for further research to verify a possibility of an involvement of glucocorticoids in the mechanisms that may provide the beneficial effects of spinal neuromodulation.

Vagus Nerve Stimulation Transiently Mitigates Chemotherapy-Induced Peripheral Neuropathy in Rats.

BackgroundChemotherapy-induced peripheral neuropathy is a severe side effect of chemotherapeutic agents. Vagus nerve stimulation attenuates neuroinflammation by activating the cholinergic anti-inflammatory pathway and thus may attenuate CIPN.MethodsAdult male Sprague-Dawley rats received intraperitoneal paclitaxel injection (2 mg/kg) every other day for a total of 4 injections. Three weeks later, the left cervical vagus nerve was exposed under general anesthesia, and the rats randomly received 20-min stimulation (1 V, 2 ms, 5 Hz, 30 s ON/5 min OFF) or sham stimulation. Heat and mechanical pain sensitivity was evaluated using Hargreaves and von Frey tests before and after treatment (n=12 per group per time point). Additionally, rats receiving paclitaxel or saline but no surgery were included. Expression of representative pro- and anti-inflammatory cytokines in dorsal root ganglia was assessed by Western blotting assays and immunohistochemistry.ResultsPaclitaxel significantly reduced the sensitivity for heat (withdrawal latency: paclitaxel 6.16 ± 0.54 s vs saline 9.93 ± 0.78 s, p<0.001) and mechanical pain (withdrawal frequency: paclitaxel 32.22 ± 15.51% vs saline 3.33 ± 4.92%, p<0.001). Compared with sham-stimulated rats, rats receiving vagus nerve stimulation had significantly higher sensitivity for heat (withdrawal latency: VNS 10.28 ± 1.15 s vs sham 6.27 ± 0.56 s, p<0.001) and mechanical pain (withdrawal frequency: VNS 10.00 ± 9.54% vs Sham 31.67 ± 18.99%, p=0.003) on +1 day, but not 7 days later (withdrawal latency: VNS 6.97 ± 1.13 s vs Sham 6.23 ± 0.79 s, p=0.080; withdrawal frequency: VNS 21.67 ± 11.93% vs Sham 23.33 ± 7.79%, p=0.689). Western blotting assays and immunohistochemistry revealed that interleukin-10 level was elevated in the dorsal root ganglia of rats receiving vagus nerve stimulation while no apparent changes in NF-κB or TNF-α levels were observed.ConclusionVagus nerve stimulation could transiently attenuate paclitaxel-induced hyperalgesia in rats. Future studies are needed to investigate whether stimulation with different protocols could achieve durable effects.

Loss of Brain-Derived Neurotrophic Factor Mediates Inhibition of Hippocampal Long-Term Potentiation by High-Intensity Sound.

Exposure to noise produces cognitive and emotional disorders, and recent studies have shown that auditory stimulation or deprivation affects hippocampal function. Previously, we showed that exposure to high-intensity sound (110dB, 1min) strongly inhibits Schaffer-CA1 long-term potentiation (LTP). Here we investigated possible mechanisms involved in this effect. We found that exposure to 110dB sound activates c-fos expression in hippocampal CA1 and CA3 neurons. Although sound stimulation did not affect glutamatergic or GABAergic neurotransmission in CA1, it did depress the level of brain-derived neurotrophic factor (BDNF), which is involved in promoting hippocampal synaptic plasticity. Moreover, perfusion of slices with BDNF rescued LTP in animals exposed to sound stimulation, whereas BDNF did not affect LTP in sham-stimulated rats. Furthermore, LM22A4, a TrkB receptor agonist, also rescued LTP from sound-stimulated animals. Our results indicate that depression of hippocampal BDNF mediates the inhibition of LTP produced by high-intensity sound stimulation.

Transcranial Current Stimulation Alters the Expression of Immune-Mediating Genes.

Despite its extensive use in clinical studies, the molecular mechanisms underlying the effects of transcranial direct current stimulation (tDCS) remain to be elucidated. We previously described subacute effects of tDCS on immune- and stem cells in the rat brain. To investigate the more immediate effects of tDCS regulating those cellular responses, we treated rats with a single session of either anodal or cathodal tDCS, and analyzed the gene expression by microarray; sham-stimulated rats served as control. Anodal tDCS increased expression of several genes coding for the major histocompatibility complex I (MHC I), while cathodal tDCS increased the expression of the immunoregulatory protein osteopontin (OPN). We confirmed the effects of gene upregulation by immunohistochemistry at the protein level. Thus, our data show a novel mechanism for the actions of tDCS on immune- and inflammatory processes, providing a target for future therapeutic studies.

Influences of anterior thalamic nucleus stimulation on neurogenesis in hippocampus of epileptic rats

Objective To investigate the influences of anterior thalamic nucleus (ANT) stimulation on neurogenesis in hippocampus of epileptic rats. Methods Thirty-two male SD rats were randomly allocated to normal-control group(n=8), control-stimulation group(n=8), sham-stimulation group(n=8) and model-stimulation group(n=8). Eqileptic SD rat model was established by using microinjection of kainic acid in hippocampal CA3 area, and administered 48 h continuous ANT stimulation in the chronic stage.Epileptic seizures were monitored and counted.The levels of Ki-67, a neurogenesis protein in hippocampus was determined by Western blot and quantitative real-time polymerase chain reaction (qRT-PCR). Results The epilepsy seizure rate was (5.9±2.2) per week in the sham-stimulation group and (2.9±1.1) per week in model-stimulation group.Compared with sham-stimulated rats, ANT stimulation reduced seizures by 50.8% (P<0.05). Western blot analysis revealed that the relative levels of Ki-67 in the hippocampus of model-stimulation group significantly higher than that of the sham-stimulation group((0.44±0.15) vs (0.19±0.73), P<0.05). qRT-PCR analysis showed that relative levels of Ki-67 mRNA in the hippocampus of model-stimulation group were significantly higher than that of the sham-stimulation group((0.45±0.10) vs (0.15±0.06), P<0.05). Conclusion Chronic ANT stimulation can promote neurogenesis in epileptic rats, which may be a principle mechanism of the beneficial effect of ANT stimulation on epilepsy. Key words: Anterior thalamic nucleus; Electrical stimulation; Neurogenesis; Epilepsy

Paradoxical augmented relapse in alcohol-dependent rats during deep-brain stimulation in the nucleus accumbens

Case reports indicate that deep-brain stimulation in the nucleus accumbens may be beneficial to alcohol-dependent patients. The lack of clinical trials and our limited knowledge of deep-brain stimulation call for translational experiments to validate these reports. To mimic the human situation, we used a chronic-continuous brain-stimulation paradigm targeting the nucleus accumbens and other brain sites in alcohol-dependent rats. To determine the network effects of deep-brain stimulation in alcohol-dependent rats, we combined electrical stimulation of the nucleus accumbens with functional magnetic resonance imaging (fMRI), and studied neurotransmitter levels in nucleus accumbens-stimulated versus sham-stimulated rats. Surprisingly, we report here that electrical stimulation of the nucleus accumbens led to augmented relapse behavior in alcohol-dependent rats. Our associated fMRI data revealed some activated areas, including the medial prefrontal cortex and caudate putamen. However, when we applied stimulation to these areas, relapse behavior was not affected, confirming that the nucleus accumbens is critical for generating this paradoxical effect. Neurochemical analysis of the major activated brain sites of the network revealed that the effect of stimulation may depend on accumbal dopamine levels. This was supported by the finding that brain-stimulation-treated rats exhibited augmented alcohol-induced dopamine release compared with sham-stimulated animals. Our data suggest that deep-brain stimulation in the nucleus accumbens enhances alcohol-liking probably via augmented dopamine release and can thereby promote relapse.

Transcranial direct current stimulation promotes the mobility of engrafted NSCs in the rat brain.

Transcranial direct current stimulation (tDCS) is used in numerous clinical studies and considered an effective and versatile add-on therapy in neurorehabilitation. To date, however, the underlying neurobiological mechanisms remain elusive. In a rat model of tDCS, we recently observed a polarity-dependent accumulation of endogenous neural stem cells (NSCs) in the stimulated cortex. Based upon these findings, we hypothesized that tDCS may exert a direct migratory effect on endogenous NSCs towards the stimulated cortex. Using noninvasive imaging, we here investigated whether tDCS may also cause a directed migration of engrafted NSCs. Murine NSCs were labeled with superparamagnetic particles of iron oxide (SPIOs) and implanted into rat striatum and corpus callosum. MRI was performed (i) immediately after implantation and (ii) after 10 tDCS sessions of anodal or cathodal polarity. Sham-stimulated rats served as control. Imaging results were validated ex vivo using immunohistochemistry. Overall migratory activity of NSCs almost doubled after anodal tDCS. However, no directed migration within the electric field (i.e. towards or away from the electrode) could be observed. Rather, an undirected outward migration from the center of the graft was detected. Xenograft transplantation induced a neuroinflammatory response that was significantly enhanced following cathodal tDCS. This inflammatory response did not impact negatively on the survival of implanted NSCs. Data suggest that anodal tDCS increases the undirected migratory activity of implanted NSCs. Since the electric field did not guide implanted NSCs over large distances, previously observed polarity-dependent accumulation of endogenous NSCs in the cortex might have originated from local proliferation. Results enhance our understanding of the neurobiological mechanisms underlying tDCS, and may thereby help to develop a targeted and sustainable application of tDCS in clinical practice.

Anxiogenic- and antidepressant-like behavior in corneally kindled rats

BackgroundAnxiety and depression affect epileptic patients much more often than individuals from the general population. We were interested in whether corneal kindling in rats, which is a model of complex partial seizures with secondary generalization, would influence animal behavior in models of anxiety and depression. MethodsKindling was achieved by transcorneal electric stimulation and fully kindled rats were used in this study. Kindled and sham-stimulated rats were subjected to the elevated plus maze and forced swim test which are believed to be predictive models for anxiety and depression in humans, respectively. ResultsKindling significantly decreased the percentage of time spent by the rats in open arms relative to time spent in open plus closed arms and it reduced immobility time in the swim test as compared with sham-stimulated rats. ConclusionsOur results suggest that corneal kindling produces antidepressant- and anxiety-like effects in rats and it may be a useful model to study epilepsy-associated anxiety.

High frequency stimulation of the subthalamic nucleus increases c-fos immunoreactivity in the dorsal raphe nucleus and afferent brain regions

High frequency stimulation (HFS) of the subthalamic nucleus (STN) is the neurosurgical therapy of choice for the management of motor deficits in patients with advanced Parkinson’s disease, but this treatment can elicit disabling mood changes. Our recent experiments show that in rats, HFS of the STN both inhibits the firing of 5-HT (5-hydroxytryptamine; serotonin) neurons in the dorsal raphe nucleus (DRN) and elicits 5-HT-dependent behavioral effects. The neural circuitry underpinning these effects is unknown. Here we investigated in the dopamine-denervated rat the effect of bilateral HFS of the STN on markers of neuronal activity in the DRN as well as DRN input regions. Controls were sham-stimulated rats. HFS of the STN elicited changes in two 5-HT-sensitive behavioral tests. Specifically, HFS increased immobility in the forced swim test and increased interaction in a social interaction task. HFS of the STN at the same stimulation parameters, increased c-fos immunoreactivity in the DRN, and decreased cytochrome C oxidase activity in this region. The increase in c-fos immunoreactivity occurred in DRN neurons immunopositive for the GABA marker parvalbumin. HFS of the STN also increased the number of c-fos immunoreactive cells in the lateral habenula nucleus, medial prefrontal cortex but not significantly in the substantia nigra. Collectively, these findings support a role for circuitry involving DRN GABA neurons, as well as DRN afferents from the lateral habenula nucleus and medial prefrontal cortex, in the mood effects of HFS of the STN.

Effect of spinal cord stimulation in a rodent model of post‐operative ileus

Post-operative ileus (POI) is a transient impairment of gastrointestinal (GI) transit that develops after abdominal surgery. The goal of this study was to investigate the effect of spinal cord stimulation (SCS) on gastric emptying and upper GI transit in a rat model of POI. All rats had an electrode placed on the dorsal surface of the spinal cord between the T(5) and T(8) segments. After recovery, gastric emptying and upper GI transit (geometric centre and head of meal) were assessed using a radiolabelled meal fed to each rat via oral gavage. In unanaesthetized rats, SCS (15, 25, 50, 100, 200 Hz, 0.2 ms at 90% motor threshold for 15 min) was performed immediately after the meal. The sham control group had no current applied. The naïve group was without POI or SCS. Gastric emptying was significantly delayed in sham-stimulated rats with POI compared with naïve controls (39.8 +/- 6.2%vs 76.5 +/- 4.9%, P < 0.001). In rats with POI that underwent SCS, there was a significant acceleration of gastric emptying to levels that resembled those of naïve controls (65.1 +/- 7.4%, P < 0.05). However, while SCS did not normalize the geometric centre and head of the meal when compared with the naïve group, it did significantly improve both parameters compared with the sham stimulation group. In summary, SCS normalizes gastric emptying and improves upper GI transit in a rodent model of POI. Further experiments are required to address the mechanism(s) by which SCS exhibits prokinetic activity.

S.07.04 Gender differences in the 5-HT1A receptor and serotonin transporter in the human brain: a PET study

significantly (p< 0.05) higher than those receiving shamstimulation. On the other hand, the ICES treatment of the PFC significantly decreased the distance traveled in cocaine-pretreated rats to a level closer to saline shamstimulated rats (P< 0.05). In some sub-regions of the VTA and the NAc, ICES treatment reversed the altered levels of GluR1 and NMDAR1 receptor subtypes mediated by repeated cocaine. For example, repeated cocaine up-regulated GluR1 levels in the anterior VTA (cocaine effect, p< 0.05), while ICES treatment of the LH down-regulated GluR1 levels in this region (LH-stimulation effect, p< 0.05), normalizing it to levels measured in saline-treated animals. Conclusion: Cocaine but not natural reward associated behavior was affected by ICES treatment. ICES treatment of the LH sensitized the response to cocaine after repeated exposure to the drug. On the other hand, ICES treatment of the PFC caused rats to work less for cocaine and desensitized the response to a cocaine challenge. Furthermore, ICES of the LH or the PFC reduced cocaineseeking behavior in a drug-free self-administration test. These effects might be mediated by reversing some of the neural adaptations in the reward system such as alterations in NMDAR1 and GluR1, which are induced by chronic cocaine exposure. Hence, we suggest a non-pharmacologic potential treatment for addiction using ICES.

Serotonin receptor binding and mRNA expression in the hippocampus of fearful amygdala-kindled rats

Amygdala kindling in rats increases fear behavior. The neural correlates of this fear are not well understood. In this experiment, we investigated the relation between serotonin receptor binding and mRNA expression and fearful behavior in amygdala-kindled rats. Rats received either 100 kindling stimulations or sham stimulations, and their fear behavior was subsequently assessed in an unfamiliar open field. Then, the rats were sacrificed and 5-HT transporter binding, 5-HT 1A and 5-HT 2A receptor binding, and 5-HT 1A mRNA expression in several brain regions was assessed. The kindled rats were significantly more fearful in the open field than the sham-stimulated rats. They also had significantly more 5-HT 1A receptor binding and mRNA expression in the dentate gyrus than the sham-simulated rats, and these increases in 5-HT 1A receptor binding and mRNA expression were significantly correlated to the increases in fear. There were no significant differences between the kindled and sham-stimulated rats in 5-HT transporter binding or 5-HT 2A receptor binding. These results suggest that alterations in 5-HT 1A receptors in the dentate gyrus may play a role in the expression of kindled fear.

Amygdala kindling increases fear responses and decreases glucocorticoid receptor mRNA expression in hippocampal regions

Amygdala kindling dramatically increases fearful behavior in rats. Because kindling-induced fear increases in magnitude as rats receive more stimulations, kindling provides an excellent model for studying the nature and neural mechanisms of fear sensitization. In the present experiment, we studied whether the development of kindling-induced fear is related to changes in glucocorticoid receptor (GR) mRNA expression in various brain regions. Rats received 20, 60 or 100 amygdala kindling stimulations or 100 sham stimulations. One day after the final stimulation, their fearful behavior was assessed in an unfamiliar open field. Then, the rats were sacrificed and their brains were processed for in situ hybridization of GR mRNA expression. We found that compared with the sham-stimulated rats, the rats that received 60 or 100 kindling stimulations were significantly more fearful in the open field and also had significantly less GR mRNA expression in the dentate gyrus and CA1 subfield of the hippocampus. Importantly, the changes in fearful behavior were significantly correlated with the changes in GR mRNA expression. These results suggest that alterations in GR mRNA expression in hippocampal regions may play a role in the development of kindling-induced fear.

Amygdala kindling decreases insulin-like growth factor-I receptor binding sites in the rat hippocampus

The neural excitability characteristic of kindling has been linked to structural alterations such as mossy fiber sprouting and synaptic reorganization within the hippocampus. Recent evidence suggests that growth factors may play a key role in kindling-related synaptic plasticity. Insulin-like growth factors-I and -II (IGF-I/-II) and insulin are structurally-related pleiotropic growth factors known to be involved in neural growth and differentiation. In the present study, we investigated the effect of kindling on [ 125I]IGF-I, [ 125I]IGF-II and [ 125I]insulin receptor binding in the hippocampus of adult rats. Our results indicate a progressive decrease in [ 125I]IGF-I (but not [ 125I]IGF-II or [ 125I]insulin) binding sites in the CA1, hilus and the granule cell layer of the kindled rats compared to sham-stimulated rats. These results, in keeping with the established neurotrophic effects of IGF-I, suggest a potential role for this growth factor in mediating the structural alterations associated with kindling.

Hippocampal involvement in the expression of kindling-induced fear in rats

Kindling dramatically increases fearful behavior in rats. Because kindling-induced fear increases in magnitude as rats receive more stimulations, kindling provides a superb opportunity to study the nature and neural mechanisms of fear sensitization. Interestingly, these changes in behavior are accompanied by increased binding to inhibitory receptors and decreased binding to excitatory receptors in the CA1 and dentate gyrus regions of the hippocampus. This led us to hypothesize that kindling-induced fear may result from an increased inhibitory tone within hippocampal circuits. To test this hypothesis, we investigated FOS protein immunoreactivity in hippocampal and amygdalar regions of kindled rats that were exposed to an unfamiliar open field. We found that FOS immunoreactivity was significantly decreased in the CA1 region, dentate gyrus, and perirhinal cortex of kindled rats compared to sham-stimulated rats. These results support our hypothesis that kindling-induced fear may be produced by inhibition within hippocampal circuits. They also suggest that neural changes within the hippocampus may be important for the sensitization of fear.

Decreased levels of neuropeptide Y 5 receptor binding sites in two experimental models of epilepsy

It has been suggested that the anticonvulsant effects of neuropeptide Y (NPY) could be mediated by the activation of Y 2 and/or Y 5 receptors. NPY Y 1 receptor levels are known to decrease and Y 2 to increase in rat models of epilepsy. By using an autoradiographic approach, we investigated whether epilepsy models (kainic acid and kindling) are also associated with changes in Y 5 receptors. Compared with naive controls, [ 125I][Leu 31, Pro 34]PYY/BIBP3226-insensitive (Y 5) binding sites in the hippocampus (strata oriens and radiatum of CA3 and CA1) and in the neocortex (superficial layers) were unchanged in sham-stimulated rats, but reduced by approximately 50% in kindled rats (seven days after the last stimulus evokes seizure), and further reduced (to approximately −90%) 1 h after a kindled seizure. Additionally, Y 5 receptor binding sites in the hippocampus and in the neocortex were unchanged 6 h after kainic acid injection, but were highly reduced at 12 and 24 h. No changes in Y 5 binding levels were found in the dentate gyrus and the pyramidal cell layer of the hippocampus. The present data suggest that changes in Y 5 receptor levels occur in epilepsy models. These changes may play a role in seizure expression and/or in the maintenance of kindling hyperexcitability.

Functional vagal input to gastric myenteric plexus as assessed by vagal stimulation-induced Fos expression.

Immunohistochemical detection of c-Fos expression was used to identify gastric myenteric plexus neurons that receive excitatory input from vagal efferent neurons activated by electrical stimulation of the cervical vagi in anesthetized rats. Vagal stimulation-induced Fos expression increased with higher pulse frequency, so that with 16 Hz (rectangular pulses of 1 mA/0.5 ms for 30 min) approximately 30% and with 48 Hz 90% of all neurons near the lesser curvature were Fos positive. In sham-stimulated rats there was no Fos expression. The percentage of Fos-activated neurons was only slightly smaller (85% with 48 Hz) near the greater curvature. Prior atropine administration (1 mg/kg ip) had little effect on vagal stimulation-induced Fos expression, and in unilaterally stimulated rats there was no Fos expression on the contralateral (noninnervated) side of the stomach, ruling out mediation by gastric motility or secretory responses. However, polysynaptic recruitment of third- and higher-order neurons cannot be ruled out completely. These results support the idea that, at least in the stomach, functional excitatory innervation of myenteric plexus neurons by the efferent vagus is profuse and widespread, refuting the notion of only a few vagal "command neurons."

Characterization of the defensive nature of kindling-induced emotionality.

Long-term amygdala kindling produces substantial changes in emotional behavior in rats. The purpose of these experiments was to determine whether kindling-induced emotionality is fundamentally defensive or aggressive in nature. In Experiment 1, amygdala-kindled rats tested as intruders in a resident-intruder paradigm preferred an active defense strategy (i.e., defensive upright stance, jump attacks), whereas the sham-stimulated rats preferred a passive defense strategy (i.e., freezing). In Experiment 2, amygdala-kindled rats explored an unfamiliar open field significantly less than did the sham-stimulated rats, and they were significantly more resistant to capture from the unfamiliar open field than were the sham-stimulated rats. In contrast, there were no significant differences between the kindled and sham-stimulated rats in resistance to capture from their home cages. These results suggest that the emotionality produced by long-term amygdala kindling is fundamentally defensive in nature.

Distinct changes in peptide YY binding to, and mRNA levels of, Y1 and Y2 receptors in the rat hippocampus associated with kindling epileptogenesis.

Electrical kindling of the rat dorsal hippocampus induced significant changes in the binding of 125I-peptide YY to Y1 and Y2 subtypes of neuropeptide Y receptors and in their mRNA levels in the area dentata as assessed by quantitative receptor autoradiography and in situ hybridization histochemistry. Binding to Y1 receptor sites decreased by 50% (p < 0.05) in the molecular layer of the stimulated dentate gyrus, 2 days after preconvulsive stage 2 and 1 week or 1 month after generalized stage 5 seizures compared with sham-stimulated rats. Binding to Y2 receptor sites increased bilaterally by 36-87% (p < 0.05) in the hilus at stage 2 and 1 week or 1 month after stage 5. No significant changes were observed after one afterdischarge or in the other hippocampal subfields or in the cortex. Y1 receptor mRNA signal decreased bilaterally by 50-64% (p < 0.01) in the granule cell layer, 6 h but not 24 h after stages 2 and 5. The Y2 receptor mRNA signal was enhanced by 283% (p < 0.01) in the stimulated granule cell layer 24 h after stage 2. At 6 and 24 h after stage 5, mRNA levels were increased both ipsilaterally (283 and 360%, respectively; p < 0.01) and contralaterally (190 and 260%, respectively; p < 0.05). No significant changes in level of either mRNA was found following one afterdischarge. These modifications, and the enhanced neuropeptide Y release previously shown in the hippocampus, suggest that kindling is associated with lasting changes in neuropeptide Y-mediated neurotransmission.