Chloral Hydrate-anesthetized Rats Research Articles

Amphetamine Promotes Cortical Up State in Part Via Dopamine Receptors.

Cortical neurons oscillate between Up and Down states during slow wave sleep and general anesthesia. Recent studies show that Up/Down oscillations also occur during quiet wakefulness. Arousal eliminates Down states and transforms Up/Down oscillations to a persistent Up state. Further evidence suggests that Up/Down oscillations are crucial to memory consolidation, whereas their transition to a persistent Up state is essential for arousal and attention. We have shown that D-amphetamine promotes cortical Up state, and the effect depends on activation of central α1A adrenergic receptors. Here, we report that dopamine also plays a role in D-amphetamine’s effect. Thus, using local-field-potential recording in the prefrontal cortex in chloral hydrate-anesthetized rats, we showed that the Up-state promoting effect of D-amphetamine was attenuated by antagonists at either D1 or D2-like dopamine receptors. The effect was also partially mimicked by co-activation of D1 and D2-like receptors. These results are consistent with the fact that D-amphetamine increases the release of both norepinephrine and dopamine. They are also in agreement with studies showing that dopamine promotes wakefulness and mediates D-amphetamine-induced emergence from general anesthesia. The effect of D-amphetamine was not mimicked, however, by activation of either D1 or D2-like receptors alone, indicating an interdependence between D1 and D2-like receptors. The dopamine/norepinephrine precursor L-DOPA also failed to promote the Up state. While more studies are needed to understand the difference between L-DOPA and D-amphetamine, our finding may provide an explanation for why L-DOPA lacks significant psychostimulant properties and is ineffective in treating attention-deficit/hyperactivity disorder.

Droxidopa alters dopamine neuron and prefrontal cortex activity and improves attention-deficit/hyperactivity disorder-like behaviors in rats

Finding alternative treatments for attention-deficit/hyperactivity disorder (ADHD) is crucial given the safety and efficacy problems of current ADHD medications. Droxidopa, also known as L-threo-dihydroxyphenylserine (L-DOPS), is a norepinephrine prodrug that enhances brain norepinephrine and dopamine levels. In this study, we used electrophysiological tests to examine effects of L-DOPS on the prefrontal cortex (PFC) and dopamine neurons in the ventral tegmental area. We also conducted behavioral tests to assess L-DOPS’ effects on ADHD-like behaviors in rats. In chloral hydrate-anesthetized rats, PFC local field potentials oscillated between the active, depolarized UP state and the hyperpolarized DOWN state. Mimicking the effect of d-amphetamine, L-DOPS, given after the peripheral amino acid decarboxylase inhibitor, benserazide (BZ), increased the amount of time the PFC spent in the UP state, indicating an excitatory effect of L-DOPS on PFC neurons. Like d-amphetamine, L-DOPS also inhibited dopamine neurons, an effect significantly reversed by the D2-like receptor antagonist raclopride. In the behavioral tests, BZ + L-DOPS improved hyperactivity, inattention and impulsive action of the adolescent spontaneously hypertensive rat (SHR/NCrl), well-validated animal model of the combined type of ADHD. BZ + L-DOPS also reduced impulsive choice and impulsive action of Wistar rats, but did not ameliorate the inattentiveness of Wistar Kyoto rats (WKY/NCrl), proposed model of the ADHD-predominantly inattentive type. In conclusion, L-DOPS produced effects on the PFC and dopamine neurons characteristic of drugs used to treat ADHD. BZ + L-DOPS ameliorated ADHD-like behaviors in rats suggesting its potential as an alternative ADHD treatment.

Effect of ulinastatin on programmed necrosis in hippocampal neurons in a rat model of global cerebral ischemia-reperfusion

Objective To evaluate the effect of ulinastatin on programmed necrosis in hippocampal neurons in a rat model of global cerebral ischemia-reperfusion (I/R). Methods Forty-eight clean-grade healthy adult male Sprague-Dawley rats, aged 8 weeks, weighing 280-320 g, were divided into 3 groups(n=16 each)using a random number table method: sham operation group(Sham group), global cerebral I/R group(I/R group)and ulinastatin group(UT group). Global cerebral I/R was produced by 4-vessel occlusion method in chloral hydrate-anesthetized rats in I/R and UT groups. Ulinastatin 100 000 U/kg was injected via the tail vein at the onset of ischemia in group UT, and the equal volume of normal saline was given instead in Sham and I/R groups.Neurological deficit score(NDS)was estimated at 6, 12 and 24 h of reperfusion.Animals were sacrificed at 24 h of reperfusion, brains were removed and the hippocampi were obtained for examination of pathological changes (with a light microscope) and for determination of the malondialdehyde (MDA) content and superoxide dismutase (SOD) activity (by spectrophotometry), and expression of receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like protein (MLKL) in hippocampal tissues (by Western blot). Results Compared with Sham group, the NDS was significantly increased at each time point, the MDA content was increased, the SOD activity was decreased, and the expression of RIPK1, RIPK3 and MLKL was up-regulated in I/R and UT groups (P<0.05). Compared with I/R group, the NDS was significantly decreased at each time point, the MDA content was decreased, the SOD activity was increased, and the expression of RIPK1, RIPK3 and MLKL was down-regulated in UT group (P<0.05). The pathological changes of hippocampi were significantly attenuated in UT group when compared with I/R group. Conclusion The mechanism by which ulinastatin ameliorates global cerebral I/R injury is related to inhibiting programmed necrosis in hippocampal neurons of rats. Key words: Trypsin inhibitors; Reperfusion injury; Hippocampus; Neurons; Necrosis

Effects of Low Doses of Ketamine on Pyramidal Neurons in Rat Prefrontal Cortex

Indirect evidence suggests that low doses of ketamine disinhibit (excite) pyramidal neurons in the prefrontal cortex (PFC). In this study, we directly examined the effect of ketamine on PFC pyramidal neurons using simultaneous single-cell and local-field-potential (LFP) recording in chloral hydrate-anesthetized rats. In all animals studied, PFC LFPs showed oscillations (0.3–1.5 Hz) between the active UP state and the relatively quiescent DOWN state, and pyramidal neurons fired preferentially during the UP state. Ketamine (1.25–20 mg/kg, i.v.) inhibited 80% of cells tested and consistently shifted PFC LFPs toward the DOWN state. The inhibitory effect of ketamine was mimicked by MK801, but not by the NR2B-selective NMDA receptor antagonist Ro25-6981. It was not blocked by the dopamine receptor antagonist fluphenazine, the GABAA receptor antagonist picrotoxinin, or the GABAB receptor antagonist CGP46381. These results are consistent with the high density of NMDA receptors expressed on PFC pyramidal neurons and our previous studies showing that blockade of NMDA receptors by ketamine inhibits dendritic NMDA receptor-mediated bursting in PFC pyramidal neurons. Thus, in addition to the previously proposed disinhibitory effect mediated through PFC interneurons, our data suggest that ketamine has an inhibitory effect on PFC pyramidal neurons. Our evidence further suggests that the effect is mediated through non-NR2B-containing NMDA receptors, independent of ketamine’s effect on dopamine and GABA transmission. Further understanding of the two opposing effects of ketamine on PFC pyramidal neurons may provide important new insights into its mechanism of action.

Effects of hydrogen-rich saline on expression of phosphor-p38MAPK during cerebral ischemia-reperfusion in rats

Objective To evaluate the effect of hydrogen-rich saline on the expression of phosphor-p38 mitogen-activated protein kinase (p-p38MAPK) during cerebral ischemia-reperfusion (I/R) in rats. Methods Seventy-two adult male Sprague-Dawley rats, weighing 220-250 g, were randomly divided into 3 groups (n = 20 each) using a random number table: sham operation group (group S), I/R group and hydrogen-rich saline group (group I/RH). Cerebral ischemia was induced in chloral hydrate-anesthetized rats by 2 h middle cerebral artery occlusion in I/R and I/RH groups.The artery was only exposed but not occluded in group S. At 3 days before operation and immediately after onset of reperfusion, hydrogen-rich saline (0.6 mmol/L) 10 ml/kg was intraperitoneally injected in group I/RH, while the equal volume of normal saline was given in S and I/R groups.Neurological deficits were blindly assessed and scored at the end of 24 h reperfusion.The animals were then sacrificed, and brains were removed for microscopic examination and for determination of the cerebral infarct size (by TTC), brain water content, cell apoptosis (by TUNEL), and expression of p38MAPk and phosphor-p38MAPK (p-p38MAPK) (by immunohistochemistry and Western blot). Apoptosis index was calculated. Results Compared with group S, neurological deficit score, apoptosis index, brain water content and cerebral infarct size were significantly increased, and the expression of p38MAPK and p-p38MAPK was up-regulated in I/R and I/RH groups.Compared with group I/R, neurological deficit score, apoptosis index, brain water content and cerebral infarct size were significantly decreased, and the expression of p38MAPK and p-p38MAPK was down-regulated in group I/RH.The pathological changes of cerebral tissues were significantly attenuated in group I/RH as compared with group I/R. Conclusion Hydrogen-rich saline can reduce cell apoptosis through inhibiting p-p38MAPK expression, thus attenuating cerebral I/R injury in rats. Key words: Hydrogen; Reperfusion injury; Brain; Mitogen-activated protein kinases

The local application of a flavonoid, (-)-epicatechin, increases the spiking of globus pallidus neurons in a dose-dependent manner and diminishes the catalepsy induced by haloperidol.

Flavonoids are natural substances obtained from plants. Most flavonoids cross the blood-brain barrier and exert a wide range of effects on the central nervous system. These actions have been attributed to the modulation of GABA-A receptors. Although motor systems in the central nervous system express a high density of GABA-A receptors, physiological studies about the effects of flavonoids on motor nuclei are scarce. Among the nuclei of the basal ganglia, the globus pallidus is potentially important for the processing of information related to movement. The electrical activity of globus pallidus neurons depends on the GABAergic fibers coming from the striatum and recurrent collateral fibers. It is known that the basal activity of the globus pallidus is modified by blocking dopaminergic receptors. In the present work, we analyzed the effects of the local application of a flavonoid, (-)-epicatechin, on the spiking of globus pallidus neurons in chloral hydrate-anesthetized rats and determined whether (-)-epicatechin applied bilaterally to the globus pallidus can modify the catalepsy induced by systemic administration of haloperidol. The results showed that (-)-epicatechin increased the basal firing of globus pallidus neurons in a dose-dependent manner and antagonized the inhibitory effect of GABA. Bilateral infusion of (-)-epicatechin to the globus pallidus diminished the catalepsy induced by haloperidol.

Increasing endocannabinoid levels in the ventral pallidum restore aberrant dopamine neuron activity in the subchronic PCP rodent model of schizophrenia.

Background:Schizophrenia is a debilitating disorder that affects 1% of the US population. While the exogenous administration of cannabinoids such as tetrahydrocannabinol is reported to exacerbate psychosis in schizophrenia patients, augmenting the levels of endogenous cannabinoids has gained attention as a possible alternative therapy to schizophrenia due to clinical and preclinical observations. Thus, patients with schizophrenia demonstrate an inverse relationship between psychotic symptoms and levels of the endocannabinoid anandamide. In addition, increasing endocannabinoid levels (by blockade of enzymatic degradation) has been reported to attenuate social withdrawal in a preclinical model of schizophrenia. Here we examine the effects of increasing endogenous cannabinoids on dopamine neuron activity in the sub-chronic phencyclidine (PCP) model. Aberrant dopamine system function is thought to underlie the positive symptoms of schizophrenia.Methods:Using in vivo extracellular recordings in chloral hydrate–anesthetized rats, we now demonstrate an increase in dopamine neuron population activity in PCP-treated rats.Results:Interestingly, endocannabinoid upregulation, induced by URB-597, was able to normalize this aberrant dopamine neuron activity. Furthermore, we provide evidence that the ventral pallidum is the site where URB-597 acts to restore ventral tegmental area activity.Conclusions:Taken together, we provide preclinical evidence that augmenting endogenous cannabinoids may be an effective therapy for schizophrenia, acting in part to restore ventral pallidal activity.

Roles of Autophagy in MPP+-Induced Neurotoxicity In Vivo: The Involvement of Mitochondria and α-Synuclein Aggregation

Macroautophagy (also known as autophagy) is an intracellular self-eating mechanism and has been proposed as both neuroprotective and neurodestructive in the central nervous system (CNS) neurodegenerative diseases. In the present study, the role of autophagy involving mitochondria and α-synuclein was investigated in MPP+ (1-methyl-4-phenylpyridinium)-induced oxidative injury in chloral hydrate-anesthetized rats in vivo. The oxidative mechanism underlying MPP+-induced neurotoxicity was identified by elevated lipid peroxidation and heme oxygenase-1 levels, a redox-regulated protein in MPP+-infused substantia nigra (SN). At the same time, MPP+ significantly increased LC3-II levels, a hallmark protein of autophagy. To block MPP+-induced autophagy in rat brain, Atg7siRNA was intranigrally infused 4 d prior to MPP+ infusion. Western blot assay showed that in vivo Atg7siRNA transfection not only reduced Atg7 levels in the MPP+-infused SN but attenuated MPP+-induced elevation in LC3-II levels, activation of caspase 9 and reduction in tyrosine hydroxylase levels, indicating that autophagy is pro-death. The immunostaining study demonstrated co-localization of LC3 and succinate dehydrogenase (a mitochondrial complex II) as well as LC3 and α-synuclein, suggesting that autophagy may engulf mitochondria and α-synuclein. Indeed, in vivo Atg7siRNA transfection mitigated MPP+-induced reduction in cytochrome c oxidase. In addition, MPP+-induced autophagy differentially altered the α-synuclein aggregates in the infused SN. In conclusion, autophagy plays a prodeath role in the MPP+-induced oxidative injury by sequestering mitochondria in the rat brain. Moreover, our data suggest that the benefits of autophagy depend on the levels of α-synuclein aggregates in the nigrostriatal dopaminergic system of the rat brain.

Impact of Prefrontal Cortex in Nicotine-Induced Excitation of Ventral Tegmental Area Dopamine Neurons in Anesthetized Rats

Systemic administration of nicotine increases dopaminergic (DA) neuron firing in the ventral tegmental area (VTA), which is thought to underlie nicotine reward. Here, we report that the medial prefrontal cortex (mPFC) plays a critical role in nicotine-induced excitation of VTA DA neurons. In chloral hydrate-anesthetized rats, extracellular single-unit recordings showed that VTA DA neurons exhibited two types of firing responses to systemic nicotine. After nicotine injection, the neurons with type-I response showed a biphasic early inhibition and later excitation, whereas the neurons with type-II response showed a monophasic excitation. The neurons with type-I, but not type-II, response exhibited pronounced slow oscillations (SOs) in firing. Pharmacological or structural mPFC inactivation abolished SOs and prevented systemic nicotine-induced excitation in the neurons with type-I, but not type-II, response, suggesting that these VTA DA neurons are functionally coupled to the mPFC and nicotine increases firing rate in these neurons in part through the mPFC. Systemic nicotine also increased the firing rate and SOs in mPFC pyramidal neurons. mPFC infusion of a non-α7 nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine blocked the excitatory effect of systemic nicotine on the VTA DA neurons with type-I response, but mPFC infusion of nicotine failed to excite these neurons. These results suggest that nAChR activation in the mPFC is necessary, but not sufficient, for systemic nicotine-induced excitation of VTA neurons. Finally, systemic injection of bicuculline prevented nicotine-induced firing alterations in the neurons with type-I response. We propose that the mPFC plays a critical role in systemic nicotine-induced excitation of VTA DA neurons.

Bursting as a source of non‐linear determinism in the firing patterns of nigral dopamine neurons

Nigral dopamine (DA) neurons in vivo exhibit complex firing patterns consisting of tonic single-spikes and phasic bursts that encode information for certain types of reward-related learning and behavior. Non-linear dynamical analysis has previously demonstrated the presence of a non-linear deterministic structure in complex firing patterns of DA neurons, yet the origin of this non-linear determinism remains unknown. In this study, we hypothesized that bursting activity is the primary source of non-linear determinism in the firing patterns of DA neurons. To test this hypothesis, we investigated the dimension complexity of inter-spike interval data recorded in vivo from bursting and non-bursting DA neurons in the chloral hydrate-anesthetized rat substantia nigra. We found that bursting DA neurons exhibited non-linear determinism in their firing patterns, whereas non-bursting DA neurons showed truly stochastic firing patterns. Determinism was also detected in the isolated burst and inter-burst interval data extracted from firing patterns of bursting neurons. Moreover, less bursting DA neurons in halothane-anesthetized rats exhibited higher dimensional spiking dynamics than do more bursting DA neurons in chloral hydrate-anesthetized rats. These results strongly indicate that bursting activity is the main source of low-dimensional, non-linear determinism in the firing patterns of DA neurons. This finding furthermore suggests that bursts are the likely carriers of meaningful information in the firing activities of DA neurons.

Effect of Sevoflurane on Functional Recovery in Animals Sustaining Systemic Circulatory Arrest

Objective: to study the effect of sevoflurane on functional recovery in animals after clinical death. Materials and methods. Experiments were carried out on male albino rats. The cardiac vascular fascicle was ligated to simulate temporary circulatory arrest. Its time was 10 minutes. Clinical death was modeled in the animals anesthetized with sevoflurane or chloral hydrate. The functional state of resuscitated animals was evaluated from the time of recovery of effective cardiac performance, spontaneous breathing, corneal reflex, and neurological deficit scores. Their elevated plus-maze behavior was examined. Results. The rats anesthetized with chloral hydrate, unlike those anesthetized with sevoflurane, showed an earlier recovery of spontaneous external breathing and corneal reflex and, in succeeding 2 days following resuscitation, less neurological deficit scores. In addition to a prompter recovery of central nervous system functions on postresuscitation day 4, the sevoflurane-anesthetized rats had a greater gain in body weight for its value on the day of clinical death modeling than the chloral hydrate-anesthetized rats. A study of their behavioral activity showed that the resuscitated rats of both groups differed from false-operated ones in a trend towards reduced number of executions, which is indicative of the higher level of rat phobic state in the postresuscitation period. No differences were observed between the groups of the resuscitated animals in all the indicators examined. Conclusion. Ten-minute clinical death modeling in sevoflurane-anesthetized rats accelerates neurological recovery and improves their general state as compared to chloral hydrate-anesthetized rats. At the same time, both anesthesia modes stop the development of the higher level of phobic state seen in the rats in the postresuscitation period. The findings do not preclude the involvement of the preconditioning properties of sevoflurane in the postresuscitation processes of brain functional recovery. Key words: circulatory arrest, sevoflurane.

Divergent activation of ventromedial and ventrolateral dopamine systems in animal models of amphetamine sensitization and schizophrenia

Aberrant dopamine-mediated behaviours are a hallmark of a number of psychiatric disorders, including substance use disorders and schizophrenia. It has been demonstrated recently that rodent models of these diseases display enhanced dopamine neuron activity throughout the ventral tegmental area (VTA). It is known, however, that the VTA is not a homogeneous structure, and that the dopamine neuron population provides discrete, topographical innervation of nucleus accumbens subregions. In addition, these ventromedial and ventrolateral dopamine systems are known to subserve complementary but distinct aspects of goal-directed behaviour. Using in-vivo extracellular recordings of identified dopamine neurons in chloral hydrate-anaesthetized rats, we examined the level of dopamine neuron population activity across the mediolateral extent of the VTA following amphetamine sensitization or gestational methylazoxymethanol acetate (MAM) treatment, a verified rodent model of schizophrenia. Here we demonstrate that both models display an augmented medial VTA-ventromedial striatal dopamine system function that correlates with the augmented locomotor response to amphetamine observed in both models. In contrast, only MAM-treated rats exhibit an increase in VTA-ventrolateral striatal dopamine system function. This latter finding is consistent with human imaging studies in schizophrenia patients. In summary, we demonstrate that, although a number of disorders involving a hyperdopaminergic state demonstrate an increase in dopamine neuron population activity, there is divergence in the exact populations of neurons affected. This distinction probably underlies the observed differences in disease symptomatology.

Neuronal projections from ventral tegmental area to forebrain structures in rat studied by manganese-enhanced magnetic resonance imaging

Manganese-enhanced magnetic resonance imaging (MEMRI) has been widely applied to trace neuronal tracts and to monitor morphological and functional responses of specific brain circuits to changes in physiological and/or environmental conditions. In this study, we traced the efferent axonal projections from ventral tegmental area (VTA) to forebrain structures, an integrating part of the reward circuit implicated in drug addiction, in rats using MEMRI. Urethane- and chloral hydrate-anesthetized rats received injection of 100 nl of 200 mM MnCl(2) solution into the right VTA. Mn(2+)-induced signal enhancements were monitored 24 h after injection. The dose of MnCl(2) injection was shown, by histological evaluation, to have minor toxic effects to the neurons in/near the injection site. Dynamic Mn(2+)-induced signal intensity changes in urethane-anesthetized rats during a 24-h period were fit to a sigmoidal function to obtain parameters slope and t(50), which describe the dynamics of apparent Mn(2+)accumulation. The results showed that most of the forebrain structures known to receive neuronal projections from the VTA, including prefrontal cortex, nucleus accumbens, globus pallidus and caudate putaman, were enhanced at 24 h after injection of MnCl(2) into the ipsilateral VTA, and anesthesia seemed have little effects on the amount of Mn(2+)being transported from the VTA to these structures.

Oscillatory firing of dopamine neurons: Differences between cells in the substantia nigra and ventral tegmental area

Neuronal oscillations have been suggested to play an important role in information processing in the brain. Using spectral analysis, we have recently shown that the repetitive burst-like firing in many dopamine (DA) neurons in the ventral tegmental area (VTA) can be described as a slow oscillation (SO) in firing rate. In this study, we examined whether DA neurons in the adjacent substantia nigra (SN) also display a SO. DA neurons were recorded extracellularly using the cells/track technique in chloral hydrate-anesthetized rats. Spectral analysis showed that firing patterns of SN DA neurons exhibited a SO similar to that observed in VTA DA neurons. The amplitude of the SO, however, was much reduced in the SN compared with that in the VTA and so was the number of DA neurons qualified as high-SO cells. In high-SO DA neurons, the amplitude of the SO was strongly correlated with the degree of bursting, and this correlation was observed in both the VTA and SN. In low-SO cells, however, the SO was more significantly correlated with the variability of firing than with firing rate and bursting. Since the generation of the SO depends on afferent inputs to DA neurons, a better understanding of its difference between the SN and VTA may provide important insights into the neural networks that control DA neurons in the two areas.

Functional Coupling between the Prefrontal Cortex and Dopamine Neurons in the Ventral Tegmental Area

Stimulation of the prefrontal cortex (PFC) has been shown to have an excitatory influence on dopamine (DA) neurons. We report here that, under nonstimulated conditions, the activity of DA neurons in the ventral tegmental area (VTA) also covaries, on a subsecond timescale, with the activity of PFC cells. Thus, in 67% of VTA DA neurons recorded in chloral hydrate-anesthetized rats, the firing of the cell displayed a slow oscillation (SO) that was highly coherent with the activity of PFC neurons. The SO was suppressed by transections immediately caudal to the PFC or by intra-PFC infusion of tetrodotoxin, suggesting that it depends on inputs derived from the PFC. Unexpectedly, the SO in most VTA DA neurons was reversed in phase relative to PFC cell activity, suggesting that at least part of PFC information is transferred to DA neurons indirectly through inhibitory relay neurons. These results, together with those reported previously, suggest that the PFC can act through multiple pathways to exert both excitatory and inhibitory influences on DA neurons. The observed functional coupling between DA and PFC neurons further suggests that these pathways not only allow a bidirectional control of DA neurons by the PFC, but also enable action potential-dependent DA release to be coordinated, on a subsecond timescale, with glutamate release from PFC terminals. Further understanding of this coordinated activity may provide important new insights into brain functions and disorders thought to involve both VTA DA and PFC neurons.

Clozapine Blocks D-Amphetamine-Induced Excitation of Dopamine Neurons in the Ventral Tegmental Area

Current antipsychotic drugs are thought to inhibit central dopamine (DA) transmission by blocking DA receptors. Here, we provide evidence that the atypical antipsychotic drug clozapine may produce part of its effect by inhibiting a subset of excitatory inputs to DA neurons. Thus, in chloral hydrate-anesthetized rats, systemic administration of D-amphetamine produced two opposing effects on DA neurons in the ventral tegmental area. Under control conditions, D-amphetamine inhibited the firing of the cell through D2-like receptors. When D2-like receptors were blocked by raclopride, D-amphetamine excited DA neurons, instead of producing no effect. The excitation, expressed as an increase in firing rate and a slow oscillation in firing pattern, was suppressed by the adrenergic alpha1 receptor antagonist prazosin, suggesting an involvement of alpha1 receptors. In rats pretreated with the typical antipsychotic drug haloperidol, D-amphetamine also excited DA neurons. However, when given after clozapine, D-amphetamine produced no significant effects. The failure of D-amphetamine to produce an excitation is not due to an incomplete blockade of D2-like receptors by clozapine because co-treatment with clozapine and raclopride also failed to enable the excitatory effect of D-amphetamine. The suggestion that clozapine inhibits the excitatory effect of D-amphetamine is further supported by the finding that clozapine, given after D-amphetamine, reliably reversed D-amphetamine-induced excitation in raclopride-treated rats. Thus, different from raclopride and haloperidol, clozapine may inhibit DA transmission through two additive mechanisms: blockade of DA receptors and inhibition of an amphetamine-sensitive, excitatory pathway that innervates DA neurons.

The CRF 1 receptor antagonist, NBI-35965, abolished the activation of locus coeruleus neurons induced by colorectal distension and intracisternal CRF in rats

Corticotropin-releasing factor (CRF) receptors have been reported to play a role in tonic colorectal distension (CRD)-induced activation of locus coeruleus (LC) neurons. We examined the influence of repeated phasic CRDs and intracisternal (ic) CRF on the spontaneous discharge rate of LC neurons in chloral hydrate-anesthetized rats and the role of CRF receptors using the nonselective CRF 1/CRF 2 antagonist, astressin, and the water-soluble CRF 1 receptor antagonist, NBI-35965. Two consecutive phasic CRDs (43.7 ± 1.1 mm Hg, 30 s each) at a 10-min interval increased LC activity to 184.9 ± 15% and 171.9 ± 12.2%, respectively. There was no difference in magnitude, onset (within 1 s), and duration (5–7 min) of the LC responses between the 1st and 2nd CRDs. CRF (300 ng/rat, ic) injected 10 min after the 2nd CRD increased LC activity to 191.1 ± 11.2%. Astressin (3 μg, ic) completely blocked the 2nd CRD- and ic CRF-induced LC activation. Neither ic vehicle nor astressin influenced basal LC neuronal activity. NBI-35965 (10 mg/kg, iv) prevented the 2nd CRD- and ic CRF-induced LC neuronal activation, while at 5 mg significantly reduced the LC response to the 2nd CRD by 80%, but did not block that of ic CRF injected 30 min later. These findings indicate a primary role of brain CRF interacting with CRF 1 receptors in mediating the activation of LC neurons in response to a phasic CRD within the nociceptive range (>40 mm Hg). This activation may have relevance to irritable bowel syndrome characterized by lower pain threshold to CRD and hypervigilance to colonic input.

Influence of insulin on the microvascular response to inflammatory mediators in neonatal streptozotocin diabetic rats

To investigate the effect of insulin on microvascular responses to inflammatory mediators in a model of type 2 diabetes mellitus. We used the neonatal streptozotocin (n-STZ)-induced diabetes model. Diabetes was induced in male newborn (2-day-old) Wistar rats through STZ administration. Experiments were performed 10-12 weeks later. Rats were divided into control (sham-injected) and study (n-STZ) groups. Using a closed-circuit video camera coupled to a microscope, changes in mesenteric arteriolar and venular diameters induced by topical application of the inflammatory mediators histamine, bradykinin and platelet-activating factor were assessed in chloral hydrate-anesthetized rats. The n-STZ rats received NPH insulin s.c. for either 4 h or 12 days. Impaired arteriole and venule responses to the inflammatory mediators tested were observed in n-STZ rats. Both acute and chronic insulin treatment corrected the alterations. We conclude that insulin is beneficial, restoring microvascular reactivity to inflammatory mediators in type 2 diabetes.

Stimulation of serotonin2C receptors blocks the hyperactivation of midbrain dopamine neurons induced by nicotine administration.

In vivo electrophysiological techniques were used to study the effect of nicotine on the basal activity of dopamine (DA)-containing neurons in the substantia nigra pars compacta (SNc) and the ventral tegmental area (VTA) of chloral hydrate-anesthetized rats. Acute i.v. injections of nicotine (25-400 microg/kg) caused a dose-dependent increase of the firing rate and the bursting activity of DA neurons both in the SNc and the VTA. Repeated daily injection of nicotine (1 mg/kg i.p.) for 10 consecutive days did not cause any significant change in the basal activity of DA neurons in the SNc and the VTA. Acute challenge with nicotine (25-400 microg/kg i.v.) in animals treated repeatedly with this drug caused a dose-related excitation of DA neurons in both areas. To test the hypothesis that stimulation of 5-hydroxytryptamine (5-HT, serotonin)(2C) receptors could affect nicotine-induced stimulation of DA neuronal activity, the selective 5-HT(2C) receptor agonist RO 60-0175 was used. Pretreatment with 100 microg/kg i.v. (S)-2-(chloro-5-fluoro-indo-l-yl)-l-methylethylamine 1:1 C(4)H(4)O(4) (RO 60-0175) prevented the enhancement in DA neuronal firing rate elicited by acute nicotine (25-400 microg/kg i.v.) in the SNc of both drug naive and chronically treated rats but was devoid of any significant effect in the VTA. Moreover, the dose of 300 microg/kg i.v. RO 60-0175 significantly reduced the stimulatory effect of VTA DA neurons induced by acute challenge with nicotine (25-400 microg/kg i.v.) both in drug naive and chronically treated rats. It is concluded that selective activation of 5-HT(2C) receptors can block the stimulatory action of nicotine on midbrain DA neuronal activity.

Kynurenate in the pontine reticular formation inhibits acoustic and trigeminal nucleus-evoked startle, but not vestibular nucleus-evoked startle

The startle reflex is elicited by acoustic, trigeminal or vestibular stimulation, or by combinations of these stimuli. Acoustic startle is mediated largely by ibotenate-sensitive neurons in the ventrocaudal pontine reticular formation (PnC). In these studies we tested whether startle elicited by stimulation of different modalities is affected by infusion of the non-selective glutamate antagonist, kynurenate, into the PnC. In awake rats, startle responses evoked by either acoustic or spinal trigeminal nucleus stimulation were inhibited by kynurenate, but not saline, infusions, with the most effective placements nearest PnC. In chloral hydrate-anesthetized rats, kynurenate in the PnC reduced trigeminal nucleus-evoked hindlimb EMG responses, but not vestibular nucleus-evoked startle. Kynurenate in the vestibular nucleus had no effect on trigeminal nucleus-evoked startle. These results indicate that trigeminal nucleus stimulation evokes startle largely through glutamate receptors in the PnC, similarly to acoustic startle, but vestibular nucleus-evoked startle is mediated through other pathways, such as the vestibulospinal tract.