Effects of Metabotropic Glutamate Receptor Stimulation on Blood–Brain Barrier Permeability During Focal Cerebral Ischemia

This investigation was performed to evaluate whether ACPD [(1S, 3R)-1-aminocyclopentane-1, 3-dicarboxylic acid], a metabotropic glutamate receptor agonist, would enhance the degree of increase in blood-brain barrier (BBB) permeability caused by focal cerebral ischemia. In this study, male Wistar rats were placed in control (n = 7) and ACPD (n = 7) groups under isoflurane anesthesia. Twenty minutes after middle cerebral artery (MCA) occlusion, patches of 10(-5) M ACPD or normal saline were placed on the ischemic cortex (IC) for a period of 40 min. Patches were changed every 10 min. One hour after MCA occlusion, BBB permeability was determined by measuring the transfer coefficient (Ki) of [alpha-14C] aminoisobutyric acid. There were no statistical differences in systemic blood pressures and heart rates between these groups. Blood gases were within normal limits. In the control group, the Ki of ischemic cortex (IC) was 2.1 times that of the contralateral cortex (CC) (3.7+/-0.9 vs. 1.8+/-0.3 microl/g/min). In the ACPD group, the Ki of the IC was 3.3 times that of the CC (5.0+/-0.7 vs. 1.5+/-0.4 microl/g/min). The increase in Ki of the ACPD group in the ischemic cortex was significantly greater than that in the control group. There was no significant difference in the Ki of the CC between these groups. Our data suggest that activation metabotropic glutamate receptors in the cortex can further augment the increase in BBB permeability caused by focal ischemia.     

Changes in potentiation and timing of sensorimotor transmission after adaptation of a postsynaptic transient outward current in a simulated cortical neuron

How adaptation of a postsynaptic transient outward current might affect the efficacy of sensorimotor transmission was investigated. The transmission signals that were studied were a 5 ms conditioned stimulus (CS) and a 60 ms US drawn from intracellularly recorded, depolarizing postsynaptic potentials (PSPs) elicited in pyramidal neurons of the cat motor cortex by a click CS and a glabella tap US, respectively. SPICE, a program used to analyze electrical circuits, was used to simulate the cortical neuron containing the adaptive outward current. Changes in the magnitude and latency of rise to firing threshold of the PSPs were compared i) after presynaptic augmentation of a CS input in the absence of an adaptive postsynaptic current and ii) after decreasing the magnitude of an adaptive postsynaptic current that was rapidly activated by depolarization. Effects of short (6 ms) and long (24 ms) inactivation time constants of the postsynaptic current were also studied. In both presynaptic adaptation and postsynaptic adaptation, the potentiation of the magnitude of the CS-induced PSP was similar, with the latency to threshold being reduced by < or = 1 ms in both cases. The effects on the US PSP differed. Presynaptic adaptation affecting the CS had no effect on the US. Adaptation of the CS by a postsynaptic outward current with a 6 ms inactivation time constant, reduced the latency to threshold of an EPSP from a nearby US synapse by up to 6 ms by augmenting the initial portion of the slowly rising US-induced PSP. Adaptation of a postsynaptic current with a 24 ms inactivation time constant reduced the latency of response to the US PSP by up to 16 ms. When the US synapse was relocated to the soma, the reduction in US latency caused by adaptation of the outward current at the CS synapse was reduced by up to one half. The latency of slowly rising components of integrated synaptic responses to compound CSs of > 5 ms duration from multiple synaptic inputs would be expected to show reductions corresponding to those of the US. We conclude that potentiation of synaptic transmission by adaptation of a postsynaptic outward current can result in reductions of latency of sensorimotor transmission that can significantly affect the timing and accuracy of controlled motor tasks. These effects depend significantly on the locations of the synaptic inputs within the cell.     

Extension of the CS past the US can facilitate conditioning of the rabbit's nictitating membrane response

Five experiments were conducted in which the onset of a tone conditioned stimulus (CS) preceded the unconditioned stimulus (US) by 500 ms. Across experiments, the offset of the CS was extended past the offset of the US by values ranging from 0 ms to 40000 ms. Extensions of the CS of 2000 ms or greater produced acquisition of a conditioned response (CR) that was as fast or faster than in the no-extension condition (0 ms). While extension of a forward tone CS after the US enhanced excitatory conditioning, insertion of another CS (light) in a purely backward relationship with the US passed only a retardation test, indicative of latent inhibition, and not a summation test needed for conditioned inhibition. The results add to the evidence that excitatory and inhibitory processes are both engaged following US offset. Alternative theories of CS processing are discussed.     

Genetic variation in brain-derived neurotrophic factor and human fear conditioning

Brain-derived neurotrophic factor (BDNF) has been implicated in hippocampal-dependent learning processes, and carriers of the Met allele of the Val66Met BDNF genotype are characterized by reduced hippocampal structure and function. Recent nonhuman animal work suggests that BDNF is also crucial for amygdala-dependent associative learning. The present study sought to examine fear conditioning as a function of the BDNF polymorphism. Fifty-seven participants were genotyped for the BDNF polymorphism and took part in a differential-conditioning paradigm. Participants were shocked following a particular conditioned stimulus (CS+) and were also presented with stimuli that ranged in perceptual similarity to the CS+ (20, 40 or 60% smaller or larger than the CS+). The eye blink component of the startle response was measured to quantify fear conditioning; post-task shock likelihood ratings for each stimulus were also obtained. All participants reported that shock likelihood varied with perceptual similarity to the CS+ and showed potentiated startle in response to CS ± 20% stimuli. However, only the Val/Val group had potentiated startle responses to the CS+. Met allele carrying individuals were characterized by deficient fear conditioning – evidenced by an attenuated startle response to CS+ stimuli. Variation in the BDNF genotype appears related to abnormal fear conditioning, consistent with nonhuman animal work on the importance of BDNF in amygdala-dependent associative learning. The relation between genetic variation in BDNF and amygdala-dependent associative learning deficits is discussed in terms of potential mechanisms of risk for psychopathology.     

Postnatal development of heart rate patterns elicited by an aversive CS and US in cats

Heart rate and motor responses were recorded in cats of different ages during classical conditioning. A deceleratory-acceleratory heart rate pattern observed during the CS-US interval in one- and four-week-old kittens is an alpha conditioned response, a potentiated original response to the CS. At eight weeks of age two new distinct patterns of pure acceleration or pure deceleration are acquired during conditioning and in the absence of motor learning. At 12 weeks of age and in adult subjects, heart rate patterns during the CS-US interval become more complex and conditioned motor responses can be observed. A covariance of HR acceleration and motor responses during the CS-US interval is absent in eight-week-old subjects, but quite high in 12-week-old subjects and adult cats. The data are interpreted as suggesting separate elicitatory mechanisms of HR and motor responses which may show synchrony later in ontogeny.     

Postnatal development of respiratory and vocal responses during aversive classical conditioning in cats

Respiratory rate, respiration amplitude, and vocal responses were recorded in cats of different ages during classic conditioning. Vocal responses to the conditional stimulus (CS) appeared first in 8-week-old kittens, and became prominent at older ages. An increase in respiration rate occurred after the onset of the CS in cats of all ages. Similarly, the decrease of respiration amplitude was observed at all ages, but only in 8-week-old and older subjects did this response resemble an adult pattern. In 4-week-old kittens the response was characterized by an early and brief peak, suggesting an alpha conditional response (CR). Clear and significant discrimination between a warning and a safety signal was present in both respiratory and vocal responses after the age of 8 weeks.     

Changes in potentiation and timing of sensorimotor transmission after adaptation of a postsynaptic transient outward current in a simulated cortical neuron

How adaptation of a postsynaptic transient outward current might affect the efficacy of sensorimotor transmission was investigated. The transmission signals that were studied were a 5 ms conditioned stimulus (CS) and a 60 ms US drawn from intracellularly recorded, depolarizing postsynaptic potentials (PSPs) elicited in pyramidal neurons of the cat motor cortex by a click CS and a glabella tap US, respectively. SPICE, a program used to analyze electrical circuits, was used to simulate the cortical neuron containing the adaptive outward current. Changes in the magnitude and latency of rise to firing threshold of the PSPs were compared i) after presynaptic augmentation of a CS input in the absence of an adaptive postsynaptic current and ii) after decreasing the magnitude of an adaptive postsynaptic current that was rapidly activated by depolarization. Effects of short (6 ms) and long (24 ms) inactivation time constants of the postsynaptic current were also studied. In both presynaptic adaptation and postsynaptic adaptation, the potentiation of the magnitude of the CS-induced PSP was similar, with the latency to threshold being reduced by " 1 ms in both cases. The effects on the US PSP differed. Presynaptic adaptation affecting the CS had no effect on the US. Adaptation of the CS by a postsynaptic outward current with a 6 ms inactivation time constant, reduced the latency to threshold of an EPSP from a nearby US synapse by up to 6 ms by augmenting the initial portion of the slowly rising US-induced PSP. Adaptation of a postsynaptic current with a 24 ms inactivation time constant reduced the latency of response to the US PSP by up to 16 ms. When the US synapse was relocated to the soma, the reduction in US latency caused by adaptation of the outward current at the CS synapse was reduced by up to one half. The latency of slowly rising components of integrated synaptic responses to compound CSs of > 5 ms duration from multiple synaptic inputs would be expected to show reductions corresponding to those of the US. We conclude that potentiation of synaptic transmission by adaptation of a postsynaptic outward current can result in reductions of latency of sensorimotor transmission that can significantly affect the timing and accuracy of controlled motor tasks. These effects depend significantly on the locations of the synaptic inputs within the cell.     

Cyclothiazide Unmasks an AMPA-Evoked Release of Arachidonic Acid from Cultured Striatal Neurones

Abstract: The joint, but not independent, activation of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and metabotropic glutamate receptors induces liberation of arachidonic acid from cultured mouse striatal neurones. We examined whether blocking AMPA receptor desensitisation with cyclothiazide would modify this response. Cyclothiazide strongly potentiated the combined AMPA/(1 S ,3 R )-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD)-evoked release of arachidonic acid (EC₅₀ of ∼7 µ M ) but did not modulate the basal, ACPD, or NMDA response. The enhanced liberation of arachidonic acid, observed in the presence of cyclothiazide, was due to the appearance of a genuine AMPA response that was independent of an associative activation of metabotropic receptors. The potentiated and nonpotentiated AMPA responses were inhibited by both competitive [2,3-dihydroxy-6-nitro-7-sulphamoylbenzo( f )quinoxaline] and 2,3-benzodiazepine noncompetitive (GYKI 53655 and GYKI 52466) receptor antagonists. Cyclothiazide was equally effective at potentiating the AMPA response in either the presence or absence of glucose, suggesting that the increased glutamate-evoked arachidonic acid release observed in these cells under conditions of glucose deprivation is not due to reduced AMPA receptor desensitisation. The enhanced liberation of arachidonic acid measured in the presence of cyclothiazide appeared to result from a large (fourfold) elevation of the AMPA-induced increase in intracellular calcium level. Therefore, an AMPA-evoked mobilisation of arachidonic acid could potentially contribute to non-NMDA receptor-mediated neurotoxicity, which has been observed in neuronal cells in the presence of cyclothiazide.     

Concomitant Pavlovian conditioning of heart rate and leg flexion responses in the rat

Pavlovian conditioning was studied in male Fischer 344 rats using tones as the conditioned stimulus (CS) and footshock as the unconditioned stimulus (UCS). Different groups of animals received (a) contiguous CS-UCS pairings with a 0.5 sec CS, (b) contiguous CS-UCS pairings with a 4.0 sec CS, or (c) a random sequence of noncontiguous tones and shocks using either a 0.5 sec or a 4.0 sec CS. Heart rate (HR) and leg flexion (LF) responses were recorded. Leg flexion conditioning occurred only in the 0.5 sec contiguous group. Decelerative HR CRs occurred only in the 4.0 sec contiguous group. Accelerative HR changes occurred in the other two groups but were significantly greater in the 0.5 sec contiguous group. These results are similar to but not identical to those obtained during eyeblink or nictitating membrane conditioning in rabbits, and suggest that the topography of the Pavlovian HR CR is dependent on the simultaneous occurrence of other classically conditioned responses.     

Changes in neuronal responses of area 7 of cat cerebral cortex to unconditioned and conditioned stimulation under the effects of external stimuli

The effects of external stimuli (ES) and feeding motivations on area-7 neuronal impulse responses (IR) elicited by unconditioned and conditioned stimulation in alert cats were investigated. In untrained cats, preliminary (by 1 sec) action of ES resulted in the disappearance of impulse responses to electrical stimulation of the forepaw (EFS) in the first (no more than five-eight) executions; the responses gradually reappeared and in subsequent executions reestablished their former magnitude. The emergence of feeding motivation excitation in the cats, elicited by food presentation, also promoted disappearance of the initial responses to EFS; these were reestablished only after the cats were quieted. In conditioned-reflex cats, the action of the ES, which had been suppressed after onset of the reflex, as well as lowering of initial feeding motivation level (caused by natural satiation of the cats or by change in quality of the reinforcing food item), led to increase in latent periods (by 50–250 msec) and prolongation of neuronal responses to the conditioning stimulus (CS); but these parameters were unchanged during performance of trained movements. Neuronal response lag time to the action of the CS was defined by ES delivery time and by level of feeding excitation in the cats. The reason for the variation of neuronal responses to unconditioned and conditioned stimulation in the parietal cortex is assumed to be associated with variation in exteroreceptive attention level of cats under the effects of stimuli and altered metabolism.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 23, No. 5, pp. 564–574, September–October, 1991.     

Effects of Metabotropic Glutamate Receptor Stimulation on Cerebral O2 Consumption and Blood Flow During Focal Cerebral Ischemia in Rats

This investigation was performed to evaluate the effects of ACPD [(1S, 3R)-1-aminocyclopentane-1,3-dicarboxylic acid], a metabotropic glutamate receptor agonist, on cerebral O₂ consumption during focal cerebral ischemia. Male Wistar rats were placed in control (n = 7) and ACPD (n = 7) groups under isoflurane anesthesia. Twenty minutes after middle cerebral artery (MCA) occlusion, gauze sponges with 10^–5 M ACPD or normal saline were placed on the ischemic cortex (IC) for a period of 40 min and were changed every 10 min. One hour after MCA occlusion, regional cerebral blood flow (rCBF) was determined using the C¹⁴-iodoantipyrine autoradiographic technique. Regional arterial and venous oxygen saturation were determined using microspectrophotometry. There were no statistical differences in vital signs, blood gases, and hemoglobin between the groups. In the control group, the cerebral blood flow and oxygen consumption of the IC were significantly lower than the contralateral cortex (rCBF: 45 ± 11 vs. 110 ± 11 ml/min/100 g, O₂ consumption: 2.9 ± 0.4 vs. 5.4 ± 1.1 ml O₂/min/100 g). ACPD did not change regional cerebral blood flow of the IC, but did significantly increase the oxygen extraction (7.8 ± 0.2 vs. 6.9 ± 0.3 ml O₂/100 ml) and oxygen consumption of the IC (4.3 ± 1.5 vs. 2.9 ± 0.4) compared to the control IC. Our data demonstrated that topical application of 10^–25 M ACPD to the ischemic area worsened cerebral O₂ balance. These data suggest that metabotropic glutamate receptors are not maximally activated during ischemia in the temporal cortex.     

Conditioned modification of viscerosensory evoked potentials during sleep and wakefulness in cats

The effects of classic conditioning on the viscerosensory evoked potentials (EPs) were studied in twenty cats during wakefulness (W), slow-wave-sleep (SWS) and paradoxical sleep (PS). Four types of the experiment were performed on four groups of animals. Weak, non-painful stimulation of the small intestine or of the left splanchnic nerve was used as conditional stimulus (CS) in all experiments. A painful or non-painful shock on the left radial nerve served as unconditional stimulus (US) which followed the CS with a delay of 500 ms. In the first and second series of experiments, the CS was paired with non-painful or painful CS during W. In the third and fourth types of experiment, weak US was used and conditioning was done during SWS or PS. The evoked responses were recorded from the primary (SI) and secondary (SII) somatosensory and associative (AS) cortex, the thalamus (VPL), hypothalamus (HPT) and dorsal hippocampus (HPC). In each experiment, the stimulus pairings resulted in a complex electrographic conditional response (CR) which included an amplitude increase of the late components of EP's (early CR) and the development of a wave of 500 ms latency (delayed CR). In the second experiment, however, a behavioural CR (limb flexion) also appeared. All these CRs proved to be extinguishable. The recall of CR established during W was successful in SWS. The traces of CS-US pairings during SWS could, however, be elicited only in SWS. Both establishment and recall of CR were unsuccessful during PS. The possible mechanism of the effects originating from an interaction of conditioning and sleep on the viscerosensory inputs of the brain are discussed.     

A glimpse of the brain transforming a sensory signal into a motor response

Averages were made of neuronal spike activity recorded successively from eight relay regions along the auditorimotor pathway of naive cats and cats conditioned to blink in response to a 70 dB click conditioned stimulus (CS). It was hypothesized that the patterns of activity could be distinguished as sensory or motor by differences in their relationship to the pattern of the acoustic CS vs that of the conditioned response (CR). If so, it was also hypothesized that the acoustic stimulus would be better expressed at early auditorimotor relays and the motor response at later relays along the pathway. To test these hypotheses, Pearson correlation coefficients were calculated between the mean patterns of unit activity at each of the auditorimotor relays and (1) the rectified sound pattern of the CS and (2) the averaged, rectified electromyographic (EMG) activity of the muscles (orbicularis oculis) that produced the CR. In both naive and conditioned cats, there were significant positive correlations between the patterns of spike activity and the sound at early relays along the auditorimotor pathway such as the cochlear nucleus and inferior colliculus. In the conditioned animals, the spike activity of later nuclei in the auditorimotor pathway, such as the rostral thalamus and the motor cortex, had the highest positive correlations with the motor response. These correlations were low in the naive animals. Thus, the mean patterns of spike activity along the auditorimotor pathway appeared to distinguish the sound from the motor response and provided a glimpse of the process supporting transformation of the CS into the incipient CR.     

A glimpse of the brain transforming a sensory signal into a motor response

Averages were made of neuronal spike activity recorded successively from eight relay regions along the auditorimotor pathway of naive cats and cats conditioned to blink in response to a 70 dB click conditioned stimulus (CS). It was hypothesized that the patterns of activity could be distinguished as sensory or motor by differences in their relationship to the pattern of the acoustic CS vs that of the conditioned response (CR). If so, it was also hypothesized that the acoustic stimulus would be better expressed at early auditorimotor relays and the motor response at later relays along the pathway. To test these hypotheses, Pearson correlation coefficients were calculated between the mean patterns of unit activity at each of the auditorimotor relays and (1) the rectified sound pattern of the CS and (2) the averaged, rectified electromyographic (EMG) activity of the muscles (orbicularis oculis) that produced the CR. In both naive and conditioned cats, there were significant positive correlations between the patterns of spike activity and the sound at early relays along the auditorimotor pathway such as the cochlear nucleus and inferior colliculus. In the conditioned animals, the spike activity of later nuclei in the auditorimotor pathway, such as the rostral thalamus and the motor cortex, had the highest positive correlations with the motor response. These correlations were low in the naive animals. Thus, the mean patterns of spike activity along the auditorimotor pathway appeared to distinguish the sound from the motor response and provided a glimpse of the process supporting transformation of the CS into the incipient CR.     

Effects of varying intensity exercise on shortening and intracellular calcium in ventricular myocytes from streptozotocin (STZ)-induced diabetic rats

This study examined the influence of two intensities of exercise on ventricular myocyte shortening and intracellular calcium in the streptozotocin (STZ)-induced diabetic rat. Animals were divided into four groups: control sedentary (CS), diabetic sedentary (DS), diabetic light exercise (DLE; 5 x 30 min/week, 9 m/min) and diabetic moderate exercise (DME; 5 x 30 min/week, 18 m/min) and the exercise programme started 2 months after STZ treatment. Time to peak (TPK) shortening was prolonged in myocytes from DS (112.1 +/- 2.5 ms) compared to CS (98.1 +/- 2.1 ms) rats and was not additionally altered by either light (117.0 +/- 2.1 ms) or moderate (115.4 +/- 2.0 ms) exercise. TPK of the Ca(2+) transient was not significantly altered by STZ treatment (69.4 +/- 2.4 ms) but was prolonged by light (79.8 +/- 3.5 ms) and moderate (76.6 +/- 2.9 ms) exercise compared to CS (65.5 +/- 2.7 ms). Data from this study suggest that the chosen intensities of exercise were ineffective in modulating the dynamics of cardiac muscle contraction and reversing the deleterious effects of diabetes on heart-muscle contraction.     

Conditioned reflex changes in the intra-analyzer interaction of the afferent inputs from the lateral geniculate body and pulvinar thalami in the cerebral cortex of cats

Paired heterogeneous stimulation of the lateral geniculate body (LGB) and pulvinar (Pulv) as a conditioned stimulus of alimentary instrumental conditioned reflex (CR), resulted in a change of relations between afferent inputs from LGB and Pulv to the visual and associative cortex of cats. At stimulation of LGB preceding by 40 ms, facilitation of the response to testing Pulv stimulation observed in untrained cats, appeared only at the beginning of the learning and was suppressed by the end of elaboration, when the amplitude of the response to the conditioning LGB stimulation greatly increased. In the process of CR elaboration (in the middle of learning), Pulv stimulation preceding by 40 ms facilitated the response to the testing LGB stimulation and simultaneously increased the amplitude of the response to the conditioning Pulv stimulation.     

Classical conditioning of breathing pattern after two acquisition trials in 2-day-old mice.

The aim of the present study was to test whether breathing pattern conditioning may occur just after birth. We hypothesized that sensory stimuli signaling the resumption of maternal care after separation may trigger an arousal and/or orienting response accompanied with concomitant respiratory changes. We performed a conditioning experiment in 2-day-old mice by using an odor (lemon) as the conditioned stimulus (CS) and maternal care after 1 h without the mother as the unconditioned stimulus (US). Each pup underwent two acquisition trials, in which the CS was presented immediately before (experimental paired group, n = 30) or 30 min before (control unpaired group, n = 30) contact with the mother. Conditioning was tested by using noninvasive whole body plethysmography to measure the respiratory response to the CS for 1 min. We found significantly stronger respiratory responses to the CS in the experimental group than in the control group. In contrast, somatomotor activity did not differ significantly between groups. Our results confirm the sensitivity of breathing to conditioning and indirectly support the hypothesis that learned feedforward processes may complement feedback pathways during postnatal maturation of respiratory control.     

Metabotropic glutamate receptors inhibit microglial glutamate release

Pro-inflammatory stimuli evoke an export of glutamate from microglia that is sufficient to contribute to excitotoxicity in neighbouring neurons. Since microglia also express various glutamate receptors themselves, we were interested in the potential feedback of glutamate on this system. Several agonists of mGluRs (metabotropic glutamate receptors) were applied to primary rat microglia, and the export of glutamate into their culture medium was evoked by LPS (lipopolysaccharide). Agonists of group-II and -III mGluR ACPD [(1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid] and L-AP4 [L-(+)-2-amino-4-phosphonobutyric acid] were both capable of completely blocking the glutamate export without interfering with the production of NO (nitric oxide); the group-I agonist tADA (trans-azetidine-2,4-dicarboxylic acid) was ineffective. Consistent with the possibility of feedback, inhibition of mGluR by MSPG [(R,S)-α-2-methyl-4sulfonophenylglycine] potentiated glutamate export. As the group-II and -III mGluR are coupled to Gα_i-containing G-proteins and the inhibition of adenylate cyclase, we explored the role of cAMP in this effect. Inhibition of cAMP-dependent protein kinase [also known as protein kinase A (PKA)] by H89 mimicked the effect of ACPD, and the mGluR agonist had its actions reversed by artificially sustaining cAMP through the PDE (phosphodiesterase) inhibitor IBMX (isobutylmethylxanthine) or the cAMP mimetic dbcAMP (dibutyryl cAMP). These data indicate that mGluR activation attenuates a potentially neurotoxic export of glutamate from activated microglia and implicate cAMP as a contributor to this aspect of microglial action.     

Metabotropic Glutamate Receptor Agonists Potentiate Cyclic AMP Formation Induced by Forskolin or β-Adrenergic Receptor Activation in Cerebral Cortical Astrocytes in Culture

Abstract: The metabotropic glutamate receptor (mGluR) agonist 1-aminocyclopentane-1 S ,3 R -dicarboxylic acid (ACPD) potentiated the accumulation of cyclic AMP induced by either β-adrenergic receptor stimulation (isoproterenol) or direct activation of adenylyl cyclase (AC) with forskolin in rat cerebral cortical astrocytes grown in a defined medium. In contrast, ACPD inhibits the cyclic AMP response in astrocytes cultured in a serum-containing medium. Pharmacological characterization indicated that a group I mGluR, of which only mGluR5 is detectable in these cells, is involved in the potentiation of cyclic AMP accumulation. Potentiation was elicited by mGluR I agonists [e.g., ( R,S )-3,5-dihydroxyphenylglycine (DHPG)], but not by mGluR II or III agonists; it was pertussis toxin resistant and abolished by procedures suppressing mGluR5 function (phorbol ester pretreatment or DHPG-induced receptor down-regulation). Nevertheless, it appears that products generated through the mGluR5 transduction pathway, such as elevated [Ca²⁺]_i or activated protein kinase C (PKC), are not involved in the potentiation as it was not influenced by either the intracellular calcium chelator BAPTA-AM or the PKC inhibitor Ro 31-8220. An inhibitor of phospholipase C, U-73122, markedly attenuated mGluR5-activated phosphoinositide hydrolysis but did not significantly affect the DHPG potentiation of the cyclic AMP response. A mechanism is proposed in which the potentiating effect on AC could be mediated by free βγ complex that is liberated after the agonist-bound mGluR5 interacts with its coupled G protein.     

Phylogeny of Helicobacter felis sp. nov., Helicobacter mustelae, and related bacteria.

Strain CS1T (T = type strain) is a gram-negative, microaerophilic, urease-positive, spiral-shaped bacterium that was isolated from the gastric mucosa of a cat. Additional strains which possessed biochemical and ultrastructural characteristics similar to those of strain CS1T were isolated from the gastric mucosa of cats and dogs. The guanine-plus-cytosine content of the DNA of strain CS1T was 42.5 mol%. The 16S rRNA sequences of strain CS1T, strain DS3 (a spiral-shaped isolate from a dog), and Helicobacter mustelae were determined by direct RNA sequencing, using a modified Sanger method. These sequences were compared with the 16S rRNA sequences of Helicobacter pylori, "Flexispira rappini," Wolinella succinogenes, and 11 species of campylobacters. A dendrogram was constructed based upon sequence similarities. Strains CS1T and DS3 were very closely related (level of similarity, 99.3%). Two major phylogenetic groups were formed; one group consisted of strains CS1T and DS3, H. mustelae, H. pylori, "F. rappini," and W. succinogenes, and the other group contained the true campylobacters. The average level of similarity between members of these two groups was 84.9%. Within the first group, strains CS1T and DS3, H. pylori, and H. mustelae formed a cluster of organisms with an interspecies similarity level of 94.5%. The phylogenetic positions of W. succinogenes and "F. rappini" were just outside this cluster. On the basis of the results of this study, we believe that strains CS1T (= ATCC 49179T) and DS3 represent a new species of the genus Helicobacter, for which we propose the name Helicobacter felis.