Gamma Isoform Of Protein Kinase Research Articles

Subpopulations of PKCγ interneurons within the medullary dorsal horn revealed by electrophysiologic and morphologic approach.

Mechanical allodynia, a cardinal symptom of persistent pain, is associated with the unmasking of usually blocked local circuits within the superficial spinal or medullary dorsal horn (MDH) through which low-threshold mechanical inputs can gain access to the lamina I nociceptive output neurons. Specific interneurons located within inner lamina II (IIi) and expressing the gamma isoform of protein kinase C (PKCγ⁺) have been shown to be key elements for such circuits. However, their morphologic and electrophysiologic features are still unknown. Using whole-cell patch-clamp recordings and immunohistochemical techniques in slices of adult rat MDH, we characterized such lamina IIi PKCγ⁺ interneurons and compared them with neighboring PKCγ⁻ interneurons. Our results reveal that PKCγ⁺ interneurons display very specific activity and response properties. Compared with PKCγ⁻ interneurons, they exhibit a smaller membrane input resistance and rheobase, leading to a lower threshold for action potentials. Consistently, more than half of PKCγ⁺ interneurons respond with tonic firing to step current. They also receive a weaker excitatory synaptic drive. Most PKCγ⁺ interneurons express Ih currents. The neurites of PKCγ⁺ interneurons arborize extensively within lamina IIi, can spread dorsally into lamina IIo, but never reach lamina I. In addition, at least 2 morphologically and functionally different subpopulations of PKCγ⁺ interneurons can be identified: central and radial PKCγ⁺ interneurons. The former exhibit a lower membrane input resistance, rheobase and, thus, action potential threshold, and less PKCγ⁺ immunoreactivity than the latter. These 2 subpopulations might thus differently contribute to the gating of dorsally directed circuits within the MDH underlying mechanical allodynia.

Pathological brain plasticity and cognition in the offspring of males subjected to postnatal traumatic stress.

Traumatic stress in early-life increases the risk for cognitive and neuropsychiatric disorders later in life. Such early stress can also impact the progeny even if not directly exposed, likely through epigenetic mechanisms. Here, we report in mice that the offspring of males subjected to postnatal traumatic stress have decreased gene expression in molecular pathways necessary for neuronal signaling, and altered synaptic plasticity when adult. Long-term potentiation is abolished and long-term depression is enhanced in the hippocampus, and these defects are associated with impaired long-term memory in both the exposed fathers and their offspring. The brain-specific gamma isoform of protein kinase C (Prkcc) is one of the affected signaling components in the hippocampus. Its expression is reduced in the offspring, and DNA methylation at its promoter is altered both in the hippocampus of the offspring and the sperm of fathers. These results suggest that postnatal traumatic stress in males can affect brain plasticity and cognitive functions in the adult progeny, possibly through epigenetic alterations in the male germline.

A mutation in protein kinase C-gamma alters SNC neuron morphology and decreases synaptic vesicles in dopaminergic striatal terminals in the AS/AGU rat

A spontaneous mutation in the Albino Swiss (AS) rat has been shown to be a single point mutation (agu) in the gene coding for the gamma isoform of protein kinase C (PKC-γ). The characteristics of the mutant include movement disorders, a failure to release dopamine in the striatum and elevations of molecules such as parkin and ubiquitin in the substantia nigra pars compacta (SNC). This present study examined SNC cell bodies and dopaminergic synaptic terminals within the caudate-putamen. Cell volume and nuclear volume were reduced in the AS/AGU mutant compared to the AS control, but the volume fractions of mitochondria and rough endoplasmic reticulum were significantly higher. No Lewy bodies were present in the mutant, although microglia were found adjacent to some SNC cells. Dopaminergic terminals were identified in the caudate-putamen by electron microscopy with low-glutaraldehyde fixation and immunohistochemistry for tyrosine hydroxylase using immuno-gold visualisation. AS/AGU mutant rats had less than half of the synaptic vesicles of AS controls; this was not only true of “readily-releasable” zones adjacent to the synaptic cleft but also “storage pool” zones. The findings support the hypothesis that the initial bar to dopamine availability in the striatum is the reduced release, with nigral cell death being a later phenomenon.

Tyrosine hydroxylase and Lewy body molecules immunoreactivity in the SNC neurons of an AS/AGU mutantrat

The AS/AGU rat has a recessive single point mutation in the gene coding for the gamma isoform of protein kinase C (PKC-γ) resulting in a failure to release dopamine in the striatum and impaired movement including a staggering gait, difficulty in initiating movement and a slight whole body tremor. This study examined the levels tyrosine hydroxylase, ubiquitin and parkin in individual SNC cell bodies, there was no evidence of a reduction in tyrosine hydroxylase levels although levels of ubiquitin and parkin were elevated in the cytoplasm. The findings support the hypothesis that the initial bar to dopamine availability in the striatum is reduced release, with substantia nigra cell death being a later phenomenon.

Progesterone Prevents Nerve Injury-Induced Allodynia and Spinal NMDA Receptor Upregulation in Rats

Peripheral nerve injury-evoked neuropathic pain still remains a therapeutic challenge. Recent studies support the notion that progesterone, a neuroactive steroid, may offer a promising perspective in pain modulation. Evaluate the effect of progesterone administration on the development of neuropathic pain-associated allodynia and on the spinal expression of N-Methyl-D-Aspartate Receptor subunit 1 (NR1), its phosphorylated form (pNR1), and the gamma isoform of protein kinase C (PKCγ), all key players in the process of central sensitization, in animals subjected to a sciatic nerve constriction. Male Sprague-Dawley rats were subjected to a sciatic nerve single ligature constriction and treated with daily subcutaneous injections of progesterone (16 mg/kg) or vehicle. The development of hindpaw mechanical and thermal allodynia was assessed using the von Frey and Choi tests, respectively. Twenty two days after injury, the number of neuronal profiles exhibiting NR1, pNR1, or PKCγ immunoreactivity was determined in the dorsal horn of the lumbar spinal cord. Injured animals receiving progesterone did not develop mechanical allodynia and showed a significantly lower number of painful responses to cold stimulation. In correlation with the observed attenuation of pain behaviors, progesterone administration significantly reduced the number of NR1, pNR1, and PKCγ immunoreactive neuronal profiles. Our results show that progesterone prevents allodynia in a rat model of sciatic nerve constriction and reinforce its role as a potential treatment for neuropathic pain.

Glycine inhibitory dysfunction induces a selectively dynamic, morphine-resistant, and neurokinin 1 receptor- independent mechanical allodynia.

Dynamic mechanical allodynia is a widespread and intractable symptom of neuropathic pain for which there is a lack of effective therapy. We recently provided a novel perspective on the mechanisms of this symptom by showing that a simple switch in trigeminal glycine synaptic inhibition can turn touch into pain by unmasking innocuous input to superficial dorsal horn nociceptive specific neurons through a local excitatory, NMDA-dependent neural circuit involving neurons expressing the gamma isoform of protein kinase C. Here, we further investigated the clinical relevance and processing of glycine disinhibition. First, we showed that glycine disinhibition with strychnine selectively induced dynamic but not static mechanical allodynia. The induced allodynia was resistant to morphine. Second, morphine did not prevent the activation of the neural circuit underlying allodynia as shown by study of Fos expression and extracellular-signal regulated kinase phosphorylation in dorsal horn neurons. Third, in contrast to intradermal capsaicin injections, light, dynamic mechanical stimuli applied under disinhibition did not produce neurokinin 1 (NK1) receptor internalization in dorsal horn neurons. Finally, light, dynamic mechanical stimuli applied under disinhibition induced Fos expression only in neurons that did not express NK1 receptor. To summarize, the selectivity and morphine resistance of the glycine-disinhibition paradigm adequately reflect the clinical characteristics of dynamic mechanical allodynia. The present findings thus reveal the involvement of a selective dorsal horn circuit in dynamic mechanical allodynia, which operates through superficial lamina nociceptive-specific neurons that do not bear NK1 receptor and provide an explanation for the differences in the pharmacological sensitivity of neuropathic pain symptoms.

How glycine inhibitory dysfunction turns touch into pain

Event Abstract Back to Event How glycine inhibitory dysfunction turns touch into pain Daniel Voisin1*, L S. Miraucourt1 and R. Dallel1 1 University Bordeaux 2, Integrative physiology of neuroendocrine systems, France Dynamic mechanical allodynia is a widespread and intractable symptom of neuropathic pain for which there is a lack of effective therapy. During tactile allodynia, activation of the sensory fibers, which normally detect touch, elicits pain. We recently provided a novel perspective on the mechanisms of this symptom by showing that a simple switch in trigeminal glycinergic synaptic inhibition can turn touch into pain by unmasking innocuous input to superficial dorsal horn nociceptive specific neurons through a local, excitatory, NMDA-dependent neural circuit involving neurons expressing the gamma isoform of protein kinase C. Glycinergic disinhibition selectively induced a morphine resistant, dynamic, but not static, mechanical allodynia, which output operated through superficial lamina nociceptive neurons lacking the neurokinin 1 receptor. Our findings thus revealed the involvement of a selective dorsal horn circuit in dynamic mechanical allodynia. In addition, we found that after removal of glycinergic inhibition, innocuous input also activated astrocytes that drove the neural circuit through D-serine release. Our data thus also fuels the emerging concept that astrocytes are dynamic partners of pain signaling. Conference: 3rd Mediterranean Conference of Neuroscience , Alexandria, Egypt, 13 Dec - 16 Dec, 2009. Presentation Type: Oral Presentation Topic: Symposium 04 – Dis-inhibition processes in pain sensitization Citation: Voisin D, Miraucourt LS and Dallel R (2009). How glycine inhibitory dysfunction turns touch into pain. Front. Neurosci. Conference Abstract: 3rd Mediterranean Conference of Neuroscience . doi: 10.3389/conf.neuro.01.2009.16.015 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 18 Nov 2009; Published Online: 18 Nov 2009. * Correspondence: Daniel Voisin, University Bordeaux 2, Integrative physiology of neuroendocrine systems, Bordeaux, France, daniel.voisin@inserm.fr Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Daniel Voisin L S Miraucourt R. Dallel Google Daniel Voisin L S Miraucourt R. Dallel Google Scholar Daniel Voisin L S Miraucourt R. Dallel PubMed Daniel Voisin L S Miraucourt R. Dallel Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Glycine Inhibitory Dysfunction Turns Touch into Pain through PKCgamma Interneurons

Dynamic mechanical allodynia is a widespread and intractable symptom of neuropathic pain for which there is a lack of effective therapy. During tactile allodynia, activation of the sensory fibers which normally detect touch elicits pain. Here we provide a new behavioral investigation into the dynamic component of tactile allodynia that developed in rats after segmental removal of glycine inhibition. Using in vivo electrophysiological recordings, we show that in this condition innocuous mechanical stimuli could activate superficial dorsal horn nociceptive specific neurons. These neurons do not normally respond to touch. We anatomically show that the activation was mediated through a local circuit involving neurons expressing the gamma isoform of protein kinase C (PKCγ). Selective inhibition of PKCγ as well as selective blockade of glutamate NMDA receptors in the superficial dorsal horn prevented both activation of the circuit and allodynia. Thus, our data demonstrates that a normally inactive circuit in the dorsal horn can be recruited to convert touch into pain. It also provides evidence that glycine inhibitory dysfunction gates tactile input to nociceptive specific neurons through PKCγ-dependent activation of a local, excitatory, NMDA receptor-dependent, circuit. As a consequence of these findings, we suggest that pharmacological inhibition of PKCγ might provide a new tool for alleviating allodynia in the clinical setting.

Deficits in the mid‐brain raphe nuclei and striatum of the AS/AGU rat, a protein kinase C‐γ mutant

The AS/AGU rat carries a recessive mutation (agu) in the gene coding for the gamma isoform of protein kinase C. The rat is characterized by disordered locomotion and progressive dysfunction of the nigrostriatal dopaminergic (DA) system. This dysfunction begins with a failure to release DA within the striatum and culminates in cell loss within the substantia nigra pars compacta. The present study examines another midbrain aminergic system with input to the basal ganglia, the serotonergic (5-HT) raphe-striatal system originating in the dorsal raphe nucleus. By 3 months after birth, there is a very substantial reduction in the extracellular levels of 5-HT in the dorsal caudate-putamen of the mutants compared with controls (c. 70%). This is accompanied by a proportional increase in the levels of the 5-HT metabolite 5-hydroxyindole acetic acid (5-HIAA). At a later age, there are reductions in whole tissue 5-HT (and increases in 5-HIAA) in both the striatum and the region containing the dorsal raphe nucleus, as well as numbers of 5-HT-immunoreactive cells in the dorsal raphe nucleus. The median raphe appears to be unaffected. The results are seen in terms of an initial dysfunction in transmitter release leading to cell death, perhaps through the formation of free radicals or neurotoxins.

Uncompetitive antagonists of the N-methyl-D-aspartate (NMDA) receptors alter the mRNA expression of proteins associated with the NMDA receptor complex.

N-methyl-D-aspartate (NMDA) receptor function appears to be under complex control during physiological and pharmacological states. We have investigated the effects of acute administration of uncompetitive NMDA receptor antagonists on mRNA levels of NMDA receptor subunits and on molecules known to cluster or phosphorylate the receptor utilizing in situ hybridization on rat brain sections. A high dose (5 mg/kg; 4 hr) of dizocilpine (MK-801) decreased mRNA levels of NMDA receptor subunits NR2C and NR2B in the entorhinal and parietal cortices, respectively. MK-801 increased mRNA levels of synapse-associated protein-90/postsynaptic density-95 (SAP90/PSD-95) and a gamma-isoform of protein kinase C (PKCgamma) in cortical regions. Synapse-associated protein-97 (SAP97) mRNA levels were increased in the entorhinal cortex layer III after MK-801 or after relatively high doses of other uncompetitive NMDA receptor antagonists: phencyclidine (15 mg/kg; 6 hr) and memantine (50 mg/kg; 6 hr). Memantine also increased SAP97 mRNA expression in other cortical regions, but this effect was not observed with MK-801 or phencyclidine. NMDA receptor uncompetitive antagonists alter the expression of multiple receptor components and such events may ultimately play a role in adaptation or toxic responses.

Interplay between the gamma isoform of PKC and calcineurin in regulation of vulnerability to focal cerebral ischemia.

Protein phosphorylation and dephosphorylation mediated by protein kinases and protein phosphatases, respectively, represent essential steps in a variety of vital neuronal processes that could affect susceptibility to ischemic stroke. In this study, the role of the neuron-specific gamma isoform of protein kinase C (gammaPKC) in reversible focal ischemia was examined using mutant mice in which the gene for gammaPKC was knocked-out (gammaPKC-KO). A period of 150 minutes of unilateral middle cerebral artery and common carotid artery (MCA/CCA) occlusion followed by 21.5 hours of reperfusion resulted in significantly larger (P < 0.005) infarct volumes (n = 10; 31.1+/-4.2 mm3) in gammaPKC-KO than in wild-type (WT) animals (n = 12; 22.6+/-7.4 mm3). To control for possible differences related to genetic background, the authors analyzed Balb/cJ, C57BL/6J, and 129SVJ WT in the MCA/CCA model of focal ischemia. No significant differences in stroke volume were detected between these WT strains. Impaired substrate phosphorylation as a consequence of gammaPKC-KO might be corrected by inhibition of protein dephosphorylation. To test this possibility, gammaPKC-KO mice were treated with the protein phosphatase 2B (calcineurin) inhibitor, FK-506, before ischemia. FK-506 reduced (P < 0.008) the infarct volume in gammaPKC-KO mice (n = 7; 24.6+/-4.6 mm3), but at this dose in this model, had no effect on the infarct volume in WT mice (n = 7; 20.5+/-10.7 mm3). These results indicate that gammaPKC plays some neuroprotective role in reversible focal ischemia.

Mice lacking PKC gamma exhibit decreased anxiety.

To investigate the contribution of the PKC gamma isoform of protein kinase C (PKC) in neurochemical pathways regulating anxiety, mice lacking the gene encoding PKC gamma were tested with heterozygote and wild-type littermates in three approach-avoidance tests of anxiety. Null mutant mice consistently displayed a decrease in baseline anxiety-related behaviors in the elevated plus-maze, the black/white box, and the mirrored chamber. In the elevated plus-maze, mutant mice entered the open arms significantly more often and spent more time in the open arms of the maze. In the black/white box, transitions between the compartments were greatest in the null mutant mice, and in the mirrored chamber, mutant mice were markedly less anxious with significantly decreased latencies to enter and more time spent in the chamber. Indices of locomotor activity in the mazes and tests of activity in home cages indicated that the reduced anxiety observed in the mutant mice was not due to baseline locomotor activity differences among the genotypes. These results suggest that PKC gamma be considered as one factor in the etiology of anxiety, perhaps via its post-synaptic regulation of GABAA and 5-HT2 receptors, two receptors implicated in the neurobiology of anxiety.

Second messengers, the substantia gelatinosa and injury-induced persistent pain

Although there is now unequivocal evidence that the circuitry within the substantia gelatinosa is a major contributor to the transmission and control of nociceptive messages, this was not known 35 years ago, when Pat Wall first focussed attention on this region. In addition to being the target of neurochemically distinct nociceptors, this region contains a heterogeneous population of excitatory and inhibitory interneurons. This review focuses on the contribution of second messenger systems that are found in the substantia gelatinosa. In particular the review highlights their critical contribution to the development of persistent pain conditions in the setting of tissue and nerve injury. Several of the studies used animals with deletions of genes that encode major second messenger molecules, including protein kinase A, C and nitric oxide synthase. Our laboratory has shown that mice with a deletion of the gene that encodes the gamma isoform of protein kinase C (which is almost exclusively expressed in a population of interneurons of the inner part of the substantia gelatinosa) have completely normal acute pain responses. However, the allodynia that characteristically develops after injury does not occur in these mice, particularly when it is generated by partial sciatic nerve injury. By contrast, deletion of genes that encode protein kinase A subunits only show deficits in the development of tissue inflammation-induced pain. These differences highlight the selectivity that characterizes the contribution of different second messenger molecules. Because of the restricted distribution of these molecules, it is likely that they are activated by different populations of primary afferent nociceptor and under very different conditions of injury. Understanding the circuitry within the substantia gelatinosa is thus critical to elucidating the mechanisms through which these second messenger molecules contribute to the development of persistent pain in the setting of injury.

Distinct neurochemical features of acute and persistent pain.

To address the neurochemistry of the mechanisms that underlie the development of acute and persistent pain, our laboratory has been studying mice with deletions of gene products that have been implicated in nociceptive processing. We have recently raised mice with a deletion of the preprotachykinin-A gene, which encodes the peptides substance P (SP) and neurokinin A (NKA). These studies have identified a specific behavioral phenotype in which the animals do not detect a window of "pain" intensities; this window cuts across thermal, mechanical, and chemical modalities. The lowered thermal and mechanical withdrawal thresholds that are produced by tissue or nerve injury, however, were still present in the mutant mice. Thus, the behavioral manifestations of threshold changes in nociceptive processing in the setting of injury do not appear to require SP or NKA. To identify relevant neurochemical factors downstream of the primary afferent, we are also studying the dorsal horn second messenger systems that underlie the development of tissue and nerve injury-induced persistent pain states. We have recently implicated the gamma isoform of protein kinase C (PKCgamma) in the development of nerve injury-induced neuropathic pain. Acute pain processing, by contrast, is intact in the PKCgamma-null mice. Taken together, these studies emphasize that there is a distinct neurochemistry of acute and persistent pain. Persistent pain should be considered a disease state of the nervous system, not merely a prolonged acute pain symptom of some other disease conditions.

Spinal mechanisms of acute and persistent pain

Although there is considerable information about the mechanisms through which injury stimuli produce acute pain, recent studies indicate that there are significant long-term consequences of persistent injury. Pain is exacerbated, in part, because of a reorganization of spinal cord circuitry in the setting of persistent injury. This review describes our studies of the contribution of the primary afferent neurotransmitter, substance P (SP), to these changes. By following internalization of the SP receptor in spinal cord dorsal horn neurons, we have identified the stimuli that evoke SP release and the neurons that respond to these stimuli. Importantly, based on the intensities of stimuli required to evoke internalization, we conclude that SP is only released under conditions in which severe pain would be produced, that the release can be evoked by intense stimulation of somatic and visceral tissue, and that multiple stimulus modalities are effective. We also found that the numbers of neurons that are influenced increases dramatically in the setting of inflammation. Using a knockout strategy, we have also raised mice with a deletion of the preprotachykinin-A (PPT-A) gene, which encodes for SP and neurokinin A (NKA), and have identified a specific behavioral phenotype in which the animals do not detect a window of “pain” intensities; this window cuts across stimulus modalities. These results provide an important behavioral correlate of the receptor internalization studies. On the other hand, the allodynia (lowered pain threshold) that occurs in the setting of injury was not altered in these animals. Among the factors that could underlie injury-induced allodynia are the second messenger systems that are activated in dorsal horn neurons. Our studies have recently implicated the gamma isoform of protein kinase C (PKCγ) in the development of nerve injury-induced neuropathic pain. Specifically, we found that although acute pain responses of mice with a deletion of PKCγ are not altered, partial injury to the sciatic nerve (which induces a severe thermal and mechanical allodynia in the wild type mouse) is without effect in the knockout. Furthermore, the anatomical/neurochemical reorganization that typically follows sciatic nerve section does not occur in the PKCγ mutant mice. Because the spinal cord distribution of interneurons that express PKCγ is concentrated almost exclusively in the inner part of lamina II, we believe that changes in the properties of these neurons are key to the development of nerve injury-induced neuropathic pain conditions. Taken together, these studies emphasize that persistent pain should be considered a disease state of the nervous system, not merely a symptom of some other disease conditions. In the setting of persistent injury, the nervous system undergoes dramatic changes that exacerbate and prolong the pain condition. Our studies underscore the importance of preventing the long-term changes that result from persistent injury.

Long-term potentiation persistence greater in C57BL/6 than DBA/2 mice: Predicted on basis of protein kinase C levels and learning performance

The gamma isoform of protein kinase C (γ-PKC) activity is elevated and learning is superior in the inbred C57BL/6 mouse when compared to the DBA/2 mouse strain. Given the proposed link between PKC and long-term potentiation (LTP) on the one hand and PKC and learning on the other, it was predicted that LTP persistence would be greater in C57BL/6 mouse. When suprathreshold levels of tetanic stimulation were used, similar persistent LTP was observed in both C57BL/6 and DBA/2 strains. However, when tetanus was at threshold, the response in DBA/2 mice decayed to baseline in 30 min. similar to short-term potentiation (STP). Using this same paradigm with C57BL/6 mice. LTP persisted for 4 h, the longest time tested. The time course of the results parallels those observed in rat when phorbol ester, a potent PKC activator, converts STP to LTP. The present findings thus confirm the predicted difference between the two mouse strains. Moreover, the present findings are consistent with a role for γ-PKC in LTP. Since such results call attention to the need for γ-PKC interventive procedures, the relative utility of current PKC inhibitors, null mutants and antisense methods are discussed.

Evidence against a role for metabotropic glutamate receptors in mossy fiber LTP: the use of mutant mice and pharmacological antagonists

We have used a number of approaches to address a possible role of metabotropic glutamate receptors (mGluRs) in mossy fiber long-term potentiation (LTP) in the hippocampus. We have used two types of mutant mice—one lacking the mGluR1 subtype of receptor and one lacking the gamma isoform of protein kinase C. In neither type of mouse did we find any alteration in the magnitude of mossy fiber LTP. We next examined whether mGluRs might modulate the magnitude and/or threshold for the induction of mossy fiber LTP. In these experiments we used tetani that were either just subthreshold or just suprathreshold for generating LTP. The mGluR antagonist (+)-α-methyl-4-carboxyphenylglycine [(+)MCPG] did not convert a subthreshold tetanus into a suprathreshold tetanus, nor did (+)MCPG have any effect on the small amount of LTP that was generated by a just suprathreshold tetanus. Based on our studies, we have been unable to identify a role for mGluRs in mossy fiber LTP.

Mutant mice lacking the gamma isoform of protein kinase C show decreased behavioral actions of ethanol and altered function of gamma-aminobutyrate type A receptors.

Calcium/phospholipid-dependent protein kinase (protein kinase C, PKC) has been suggested to play a role in the sensitivity of gamma-aminobutyrate type A (GABAA) receptors to ethanol. We tested a line of null mutant mice that lacks the gamma isoform of PKC (PKC gamma) to determine the role of this brain-specific isoenzyme in ethanol sensitivity. We found that the mutation reduced the amount of PKC gamma immunoreactivity in cerebellum to undetectable levels without altering the levels of the alpha, beta I, or beta II isoforms of PKC. The mutant mice display reduced sensitivity to the effects of ethanol on loss of righting reflex and hypothermia but show normal responses to flunitrazepam or pentobarbital. Likewise, GABAA receptor function of isolated brain membranes showed that the mutation abolished the action of ethanol but did not alter actions of flunitrazepam or pentobarbital. These studies show the unique interactions of ethanol with GABAA receptors and suggest protein kinase isoenzymes as possible determinants of genetic differences in response to ethanol.

The redox site of NMDA receptors controls LTP expressed by NMDA — but not by AMPA — receptors

We have used a number of approaches to address a possible role of metabotropic glutamate receptors (mGluRs) in mossy fiber long-term potentiation (LTP) in the hippocampus. We have used two types of mutant mice—one lacking the mGluR1 subtype of receptor and one lacking the gamma isoform of protein kinase C. In neither type of mouse did we find any alteration in the magnitude of mossy fiber LTP. We next examined whether mGluRs might modulate the magnitude and/or threshold for the induction of mossy fiber LTP. In these experiments we used tetani that were either just subthreshold or just suprathreshold for generating LTP. The mGluR antagonist (+)-α-methyl-4-carboxyphenylglycine [(+)MCPG] did not convert a subthreshold tetanus into a suprathreshold tetanus, nor did (+)MCPG have any effect on the small amount of LTP that was generated by a just suprathreshold tetanus. Based on our studies, we have been unable to identify a role for mGluRs in mossy fiber LTP.

The alpha1-adrenoceptors: Molecular mechanisms of activation and regulation

We have used a number of approaches to address a possible role of metabotropic glutamate receptors (mGluRs) in mossy fiber long-term potentiation (LTP) in the hippocampus. We have used two types of mutant mice—one lacking the mGluR1 subtype of receptor and one lacking the gamma isoform of protein kinase C. In neither type of mouse did we find any alteration in the magnitude of mossy fiber LTP. We next examined whether mGluRs might modulate the magnitude and/or threshold for the induction of mossy fiber LTP. In these experiments we used tetani that were either just subthreshold or just suprathreshold for generating LTP. The mGluR antagonist (+)-α-methyl-4-carboxyphenylglycine [(+)MCPG] did not convert a subthreshold tetanus into a suprathreshold tetanus, nor did (+)MCPG have any effect on the small amount of LTP that was generated by a just suprathreshold tetanus. Based on our studies, we have been unable to identify a role for mGluRs in mossy fiber LTP.