Loren J. Martin

Olfactory exposure to males, including men, causes stress and related analgesia in rodents

We found that exposure of mice and rats to male but not female experimenters produces pain inhibition. Male-related stimuli induced a robust physiological stress response that results in stress-induced analgesia. This effect could be replicated with T-shirts worn by men, bedding material from gonadally intact and unfamiliar male mammals, and presentation of compounds secreted from the human axilla. Experimenter sex can thus affect apparent baseline responses in behavioral testing.

Different immune cells mediate mechanical pain hypersensitivity in male and female mice

A large and rapidly increasing body of evidence indicates that microglia-to-neuron signaling is essential for chronic pain hypersensitivity. Using multiple approaches, we found that microglia are not required for mechanical pain hypersensitivity in female mice; female mice achieved similar levels of pain hypersensitivity using adaptive immune cells, likely T lymphocytes. This sexual dimorphism suggests that male mice cannot be used as proxies for females in pain research.

The Rat Grimace Scale: A partially automated method for quantifying pain in the laboratory rat via facial expressions

We recently demonstrated the utility of quantifying spontaneous pain in mice via the blinded coding of facial expressions. As the majority of preclinical pain research is in fact performed in the laboratory rat, we attempted to modify the scale for use in this species. We present herein the Rat Grimace Scale, and show its reliability, accuracy, and ability to quantify the time course of spontaneous pain in the intraplantar complete Freunds adjuvant, intraarticular kaolin-carrageenan, and laparotomy (post-operative pain) assays. The scales ability to demonstrate the dose-dependent analgesic efficacy of morphine is also shown. In addition, we have developed software, Rodent Face Finder®, which successfully automates the most labor-intensive step in the process. Given the known mechanistic dissociations between spontaneous and evoked pain, and the primacy of the former as a clinical problem, we believe that widespread adoption of spontaneous pain measures such as the Rat Grimace Scale might lead to more successful translation of basic science findings into clinical application.

α5GABAA Receptors Mediate the Amnestic But Not Sedative-Hypnotic Effects of the General Anesthetic Etomidate

A fundamental objective of anesthesia research is to identify the receptors and brain regions that mediate the various behavioral components of the anesthetic state, including amnesia, immobility, and unconsciousness. Using complementary in vivo and in vitro approaches, we found that GABAA receptors that contain the α5 subunit (α5GABAARs) play a critical role in amnesia caused by the prototypic intravenous anesthetic etomidate. Whole-cell recordings from hippocampal pyramidal neurons showed that etomidate markedly increased a tonic inhibitory conductance generated by α5GABAARs, whereas synaptic transmission was only slightly enhanced. Long-term potentiation (LTP) of field EPSPs recorded in CA1 stratum radiatum was reduced by etomidate in wild-type (WT) but not α5 null mutant (α5−/−) mice. In addition, etomidate impaired memory performance of WT but not α5−/− mice for spatial and nonspatial hippocampal-dependent learning tasks. The brain concentration of etomidate associated with memory impairment in vivo was comparable with that which increased the tonic inhibitory conductance and blocked LTP in vitro. The α5−/− mice did not exhibit a generalized resistance to etomidate, in that the sedative-hypnotic effects measured with the rotarod, loss of righting reflex, and spontaneous motor activity were similar in WT and α5−/− mice. Deletion of the α5 subunit of the GABAARs reduced the amnestic but not the sedative-hypnotic properties of etomidate. Thus, the amnestic and sedative-hypnotic properties of etomidate can be dissociated on the basis of GABAAR subtype pharmacology.

Suppression of hippocampal TRPM7 protein prevents delayed neuronal death in brain ischemia

Cardiac arrest victims may experience transient brain hypoperfusion leading to delayed death of hippocampal CA1 neurons and cognitive impairment. We prevented this in adult rats by inhibiting the expression of transient receptor potential melastatin 7 (TRPM7), a transient receptor potential channel that is essential for embryonic development, is necessary for cell survival and trace ion homeostasis in vitro, and whose global deletion in mice is lethal. TRPM7 was suppressed in CA1 neurons by intrahippocampal injections of viral vectors bearing shRNA specific for TRPM7. This had no ill effect on animal survival, neuronal and dendritic morphology, neuronal excitability, or synaptic plasticity, as exemplified by robust long-term potentiation (LTP). However, TRPM7 suppression made neurons resistant to ischemic death after brain ischemia and preserved neuronal morphology and function. Also, it prevented ischemia-induced deficits in LTP and preserved performance in fear-associated and spatial-navigational memory tasks. Thus, regional suppression of TRPM7 is feasible, well tolerated and inhibits delayed neuronal death in vivo.

α5GABAA Receptor Activity Sets the Threshold for Long-Term Potentiation and Constrains Hippocampus-Dependent Memory

Synaptic plasticity, which is the neuronal substrate for many forms of hippocampus-dependent learning, is attenuated by GABA type A receptor (GABAAR)-mediated inhibition. The prevailing notion is that a synaptic or phasic form of GABAergic inhibition regulates synaptic plasticity; however, little is known about the role of GABAAR subtypes that generate a tonic or persistent inhibitory conductance. We studied the regulation of synaptic plasticity by α5 subunit-containing GABAARs (α5GABAARs), which generate a tonic inhibitory conductance in CA1 pyramidal neurons using electrophysiological recordings of field and whole-cell potentials in hippocampal slices from both wild-type and null mutant mice for the α5 subunit of the GABAAR (Gabra5−/− mice). In addition, the strength of fear-associated memory was studied. The results showed that α5GABAAR activity raises the threshold for induction of long-term potentiation in a highly specific band of stimulation frequencies (10–20 Hz) through mechanisms that are predominantly independent of inhibitory synaptic transmission. The deletion or pharmacological inhibition of α5GABAARs caused no change in baseline membrane potential or input resistance but increased depolarization during 10 Hz stimulation. The encoding of hippocampus-dependent memory was regulated by α5GABAARs but only under specific conditions that generate moderate but not robust forms of fear-associated learning. Thus, under specific conditions, α5GABAAR activity predominates over synaptic inhibition in modifying the strength of both synaptic plasticity in vitro and certain forms of memory in vivo.

Reducing Social Stress Elicits Emotional Contagion of Pain in Mouse and Human Strangers.

Empathy for anothers physical pain has been demonstrated in humans [1] and mice [2]; in both species, empathy is stronger between familiars. Stress levels in stranger dyads are higher than in cagemate dyads or isolated mice [2, 3], suggesting that stress might be responsible for the absence of empathy for the pain of strangers. We show here that blockade of glucocorticoid synthesis or receptors for adrenal stress hormones elicits the expression of emotional contagion (a form of empathy) in strangers of both species. Mice and undergraduates were tested for sensitivity to noxious stimulation alone and/or together (dyads). In familiar, but not stranger, pairs, dyadic testing was associated with increased pain behaviors or ratings compared to isolated testing. Pharmacological blockade of glucocorticoid synthesis or glucocorticoid and mineralocorticoid receptors enabled the expression of emotional contagion of pain in mouse and human stranger dyads, as did a shared gaming experience (the video game Rock Band) in human strangers. Our results demonstrate that emotional contagion is prevented, in an evolutionarily conserved manner, by the stress of a social interaction with an unfamiliar conspecific and can be evoked by blocking the endocrine stress response.

Modulation of NMDA Receptors by Pituitary Adenylate Cyclase Activating Peptide in CA1 Neurons Requires Gαq, Protein Kinase C, and Activation of Src

At CA1 synapses, activation of NMDA receptors (NMDARs) is required for the induction of both long-term potentiation and depression. The basal level of activity of these receptors is controlled by converging cell signals from G-protein-coupled receptors and receptor tyrosine kinases. Pituitary adenylate cyclase activating peptide (PACAP) is implicated in the regulation of synaptic plasticity because it enhances NMDAR responses by stimulating Gαs-coupled receptors and protein kinase A (Yaka et al., 2003). However, the major hippocampal PACAP1 receptor (PAC1R) also signals via Gαq subunits and protein kinase C (PKC). In CA1 neurons, we showed that PACAP38 (1 nm) enhanced synaptic NMDA, and evoked NMDAR, currents in isolated CA1 neurons via activation of the PAC1R, Gαq, and PKC. The signaling was blocked by intracellular applications of the Src inhibitory peptide Src(40-58). Immunoblots confirmed that PACAP38 biochemically activates Src. A Gαq pathway is responsible for this Src-dependent PACAP enhancement because it was attenuated in mice lacking expression of phospholipase C β1, it was blocked by preventing elevations in intracellular Ca2+, and it was eliminated by inhibiting either PKC or cell adhesion kinase β [CAKβ or Pyk2 (proline rich tyrosine kinase 2)]. Peptides that mimic the binding sites for either Fyn or Src on receptor for activated C kinase-1 (RACK1) also enhanced NMDAR in CA1 neurons, but their effects were blocked by Src(40-58), implying that Src is the ultimate regulator of NMDARs. RACK1 serves as a hub for PKC, Fyn, and Src and facilitates the regulation of basal NMDAR activity in CA1 hippocampal neurons.

Thermal analgesic effects from weak, complex magnetic fields and pharmacological interactions.

In several experiments, robust analgesia (equivalent to about 4 mg/kg of morphine) in male rats to thermal stimuli following exposures to weak (1 microT) complex magnetic fields was explored. The analgesia occurred when patterns of magnetic fields with burst-firing-like configurations were presented for 30 min once every approximately 4 s. The analgesic effects were intensity dependent. A different frequency-modulated pattern produced analgesia more quickly. The analgesic effects following exposure to the burst-firing magnetic fields were augmented conspicuously by preinjections of morphine (4 mg/kg) or agmatine (10 mg/kg), but blocked by naloxone (1 mg/kg). The results of these experiments suggest that rational design of the temporal structure of weak magnetic fields may be a novel, inexpensive, and reliable technique for elevating thresholds to some classes of painful stimuli.

Short-term Memory Impairment after Isoflurane in Mice Is Prevented by the α5 γ-Aminobutyric Acid Type A Receptor Inverse Agonist L-655,708

Background:Memory blockade is an essential component of the anesthetic state. However, postanesthesia memory deficits represent an undesirable and poorly understood adverse effect. Inhibitory &agr;5 subunit–containing &ggr;-aminobutyric acid subtype A receptors (&agr;5GABAA) are known to play a critical role in memory processes and are highly sensitive to positive modulation by anesthetics. We postulated that inhibiting the activity of &agr;5GABAA receptors during isoflurane anesthesia would prevent memory deficits in the early postanesthesia period. Methods:Mice were pretreated with L-655,708, an &agr;5GABAA receptor–selective inverse agonist, or vehicle. They were then exposed to isoflurane for 1 h (1.3%, or 1 minimum alveolar concentration, or air-oxygen control). Then, either 1 or 24 h later, mice were conditioned in fear-associated contextual and cued learning paradigms. In addition, the effect of L-655,708 on the immobilizing dose of isoflurane was studied. Motor coordination, sedation, anxiety, and the concentration of isoflurane in the brain at 5 min, 1 h, and 24 h after isoflurane were also examined. Results:Motor and sensory function recovered within minutes after termination of isoflurane administration. In contrast, a robust deficit in contextual fear memory persisted for at least 24 h. The &agr;5GABAA receptor inverse agonist, L-655,708, completely prevented memory deficits without changing the immobilizing dose of isoflurane. Trace concentrations of isoflurane were measured in the brain 24 h after treatment. Conclusions:Memory deficits occurred long after the sedative, analgesic, and anxiolytic effects of isoflurane subsided. L-655,708 prevented memory deficit, suggesting that an isoflurane interaction at &agr;5GABAA receptors contributes to memory impairment during the early postanesthesia period.