Suppression of glucocorticoid secretion and antipsychotic drugs have similar effects on the mesolimbic dopaminergic transmission

Specific antagonists of central dopaminergic receptors constitute the major class of antipsychotic drugs (APD). Two principal effects of APD are used as criteria for the pre-clinical screening of their antipsychotic action: (i) inhibition of basal and depolarization-induced activity of mesolimbic dopaminergic neurons; (ii) antagonism of the locomotor effects of dopaminergic agonists. Given that glucocorticoid hormones in animals increase dopamine release and dopamine-mediated behaviors and that high levels of glucocorticoids can induce psychotic symptoms in humans, these experiments examined whether inhibition of endogenous glucocorticoids might have APD-like effects on mesolimbic dopaminergic transmission in rats. It is shown that suppression of glucocorticoid secretion by adrenalectomy profoundly decreased (by greater than 50%): (i) basal dopaminergic release and the release of dopamine induced by a depolarizing stimulus such as morphine (2 mg/kg, s.c.), as measured in the nucleus accumbens of freely moving animals by microdialysis; (ii) the locomotor activity induced by the direct dopaminergic agonist apomorphine. The effects of adrenalectomy were glucocorticoid specific given that they were reversed by the administration of glucocorticoids at doses within the physiological range. Despite its profound diminution of dopaminergic neurotransmission, adrenalectomy neither modified the number of mesencephalic dopaminergic neurons nor induced gliosis in the mesencephalon or in the nucleus accumbens, as shown by tyrosine hydroxylase and glial fibrillary acidic protein immunostaining. In conclusion, these findings suggest that blockade of central effects of glucocorticoids might open new therapeutic strategies of behavioral disturbances.

Activation of a heterologously expressed octopamine receptor coupled only to adenylyl cyclase produces all the features of presynaptic facilitation in Aplysia sensory neurons

Short-term behavioral sensitization of the gill-withdrawal reflex after tail stimuli in Aplysia leads to an enhancement of the connections between sensory and motor neurons of this reflex. Both behavioral sensitization and enhancement of the connection between sensory and motor neurons are importantly mediated by serotonin. Serotonin activates two types of receptors in the sensory neurons, one of which is coupled to the cAMP/protein kinase A (PKA) pathway and the other to the inositol triphosphate/protein kinase C (PKC) pathway. Here we describe a genetic approach to assessing the isolated contribution of the PKA pathway to short-term facilitation. We have cloned from Aplysia an octopamine receptor gene, Ap oa1, that couples selectively to the cAMP/PKA pathway. We have ectopically expressed this receptor in Aplysia sensory neurons of the pleural ganglia, where it is not normally expressed. Activation of this receptor by octopamine stimulates all four presynaptic events involved in short-term synaptic facilitation that are normally produced by serotonin: (i) membrane depolarization; (ii) increased membrane excitability; (iii) increased spike duration; and (iv) presynaptic facilitation. These results indicate that the cAMP/PKA pathway alone is sufficient to produce all the features of presynaptic facilitation.

Ts1Cje, a partial trisomy 16 mouse model for Down syndrome, exhibits learning and behavioral abnormalities

A mouse model for Down syndrome, Ts1Cje, has been developed. This model has made possible a step in the genetic dissection of the learning, behavioral, and neurological abnormalities associated with segmental trisomy for the region of mouse chromosome 16 homologous with the so-called “Down syndrome region” of human chromosome segment 21q22. Tests of learning in the Morris water maze and assessment of spontaneous locomotor activity reveal distinct learning and behavioral abnormalities, some of which are indicative of hippocampal dysfunction. The triplicated region in Ts1Cje, from Sod1 to Mx1, is smaller than that in Ts65Dn, another segmental trisomy 16 mouse, and the learning deficits in Ts1Cje are less severe than those in Ts65Dn. In addition, degeneration of basal forebrain cholinergic neurons, which was observed in Ts65Dn, was absent in Ts1Cje.

Epidermal trans-urocanic acid and the UV-A-induced photoaging of the skin

The premature photoaging of the skin is mediated by the sensitization of reactive oxygen species after absorption of ultraviolet radiation by endogenous chromophores. Yet identification of UV-A-absorbing chromophores in the skin that quantitatively account for the action spectra of the physiological responses of photoaging has remained elusive. This paper reports that the in vitro action spectrum for singlet oxygen generation after excitation of trans-urocanic acid mimics the in vivo UV-A action spectrum for the photosagging of mouse skin. The data presented provide evidence suggesting that the UV-A excitation of trans-urocanic acid initiates chemical processes that result in the photoaging of skin.

Phospholipase Cβ4 is specifically involved in climbing fiber synapse elimination in the developing cerebellum

Elimination of excess climbing fiber (CF)–Purkinje cell synapses during cerebellar development involves a signaling pathway that includes type 1 metabotropic glutamate receptor, Gαq, and the γ isoform of protein kinase C. To identify phospholipase C (PLC) isoforms involved in this process, we generated mice deficient in PLCβ4, one of two major isoforms expressed in Purkinje cells. PLCβ4 mutant mice are viable but exhibit locomotor ataxia. Their cerebellar histology, parallel fiber synapse formation, and basic electrophysiology appear normal. However, developmental elimination of multiple CF innervation clearly is impaired in the rostral portion of the cerebellar vermis, in which PLCβ4 mRNA is predominantly expressed. By contrast, CF synapse elimination is normal in the caudal cerebellum, in which low levels of PLCβ4 mRNA but reciprocally high levels of PLCβ3 mRNA are found. These results indicate that PLCβ4 transduces signals that are required for CF synapse elimination in the rostral cerebellum.

Hyperactivity and impaired response habituation in hyperdopaminergic mice

Abnormal dopaminergic transmission is implicated in schizophrenia, attention deficit hyperactivity disorder, and drug addiction. In an attempt to model aspects of these disorders, we have generated hyperdopaminergic mutant mice by reducing expression of the dopamine transporter (DAT) to 10% of wild-type levels (DAT knockdown). Fast-scan cyclic voltammetry and in vivo microdialysis revealed that released dopamine was cleared at a slow rate in knockdown mice, which resulted in a higher extracellular dopamine concentration. Unlike the DAT knockout mice, the DAT knockdown mice do not display a growth retardation phenotype. They have normal home cage activity but display hyperactivity and impaired response habituation in novel environments. In addition, we show that both the indirect dopamine receptor agonist amphetamine and the direct agonists apomorphine and quinpirole inhibit locomotor activity in the DAT knockdown mice, leading to the hypothesis that a shift in the balance between dopamine auto and heteroreceptor function may contribute to the therapeutic effect of psychostimulants in attention deficit hyperactivity disorder.

A second-generation vaccine protects against the psychoactive effects of cocaine

The effects of immunization with the second-generation cocaine immunoconjugate GND-keyhole limpet hemocyanin (KLH) or with the anti-cocaine mAb GNC92H2 were assessed in a model of acute cocaine-induced locomotor activity. After i.p. administration of cocaine⋅HCl (15 mg/kg), rats were tested in photocell cages, and stereotypy was rated to determine preimmunization drug response (baseline). Experimental animals were subjected to an immunization protocol with GND-KLH or treated with the mAb GNC92H2. Rats were then challenged with systemic cocaine, and their locomotor responses were again measured. Active immunization with GND-KLH produced a 76% decrease in the ambulatory measure (crossovers) in the experimental group and a 12% increase in the control group compared with baseline values. Also, stereotypic behavior was significantly suppressed in the vaccinated animals. Decreases in both measures were seen in the experimental group on two subsequent challenges. The maximum effect was observed at the time of the second challenge with a dramatic 80% decrease in crossovers. Treatment with GNC92H2 resulted in a 69% decrease in crossovers compared with baseline. This effect persisted across two additional challenges over 11 days with decreases of 46–47%. In contrast, the control group showed increases of up to 28%. Significant differences between groups were observed in the stereotypic measure in all three challenges. The results indicate that these immunopharmacotherapeutic agents have significant cocaine-blockade potential and therefore may offer an effective strategy for the treatment of cocaine abuse.

Increased mitochondrial oxidative stress in the Sod2 (+/−) mouse results in the age-related decline of mitochondrial function culminating in increased apoptosis

To determine the importance of mitochondrial reactive oxygen species toxicity in aging and senescence, we analyzed changes in mitochondrial function with age in mice with partial or complete deficiencies in the mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD). Liver mitochondria from homozygous mutant mice, with a complete deficiency in MnSOD, exhibited substantial respiration inhibition and marked sensitization of the mitochondrial permeability transition pore. Mitochondria from heterozygous mice, with a partial deficiency in MnSOD, showed evidence of increased proton leak, inhibition of respiration, and early and rapid accumulation of mitochondrial oxidative damage. Furthermore, chronic oxidative stress in the heterozygous mice resulted in an increased sensitization of the mitochondrial permeability transition pore and the premature induction of apoptosis, which presumably eliminates the cells with damaged mitochondria. Mice with normal MnSOD levels show the same age-related mitochondrial decline as the heterozygotes but occurring later in life. The premature decline in mitochondrial function in the heterozygote was associated with the compensatory up-regulation of oxidative phosphorylation enzyme activity. Thus mitochondrial reactive oxygen species production, oxidative stress, functional decline, and the initiation of apoptosis appear to be central components of the aging process.

Identification of the endogenous smooth muscle myosin phosphatase-associated kinase

Ca2+ sensitization of smooth muscle contraction involves inhibition of myosin light chain phosphatase (SMPP-1M) and enhanced myosin light chain phosphorylation. Inhibition of SMPP-1M is modulated through phosphorylation of the myosin targeting subunit (MYPT1) by either Rho-associated kinase (ROK) or an unknown SMPP-1M-associated kinase. Activated ROK is predominantly membrane-associated and its putative substrate, SMPP-1M, is mainly myofibrillar-associated. This raises a conundrum about the mechanism of interaction between these enzymes. We present ZIP-like kinase, identified by “mixed-peptide” Edman sequencing after affinity purification, as the previously unidentified SMPP-1M-associated kinase. ZIP-like kinase was shown to associate with MYPT1 and phosphorylate the inhibitory site in intact smooth muscle. Phosphorylation of ZIP-like kinase was associated with an increase in kinase activity during carbachol stimulation, suggesting that the enzyme may be a terminal member of a Ca2+ sensitizing kinase cascade.

Mice transgenically overexpressing sulfonylurea receptor 1 in forebrain resist seizure induction and excitotoxic neuron death

The ability of the sulfonylurea receptor (SUR) 1 to suppress seizures and excitotoxic neuron damage was assessed in mice transgenically overexpressing this receptor. Fertilized eggs from FVB mice were injected with a construct containing SUR cDNA and a calcium-calmodulin kinase IIα promoter. The resulting mice showed normal gross anatomy, brain morphology and histology, and locomotor and cognitive behavior. However, they overexpressed the SUR1 transgene, yielding a 9- to 12-fold increase in the density of [3H]glibenclamide binding to the cortex, hippocampus, and striatum. These mice resisted kainic acid-induced seizures, showing a 36% decrease in average maximum seizure intensity and a 75% survival rate at a dose that killed 53% of the wild-type mice. Kainic acid-treated transgenic mice showed no significant loss of hippocampal pyramidal neurons or expression of heat shock protein 70, whereas wild-type mice lost 68–79% of pyramidal neurons in the CA1–3 subfields and expressed high levels of heat shock protein 70 after kainate administration. These results indicate that the transgenic overexpression of SUR1 alone in forebrain structures significantly protects mice from seizures and neuronal damage without interfering with locomotor or cognitive function.

Tumor necrosis factor-related apoptosis-inducing ligand in T cell development: Sensitivity of human thymocytes

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is a recently identified member of the tumor necrosis factor cytokine superfamily. TRAIL has been shown to induce apoptosis in various tumor cell lines, whereas most primary cells seem to be resistant. These observations have raised considerable interest in the use of TRAIL in tumor therapy. Yet little is known about the physiological function of TRAIL. This is particularly the case in the immune system, where TRAIL has been suggested by some to be involved in target cell killing and lymphocyte death. We have developed a panel of mAbs and soluble proteins to address the role of TRAIL in lymphocyte development. These studies demonstrate activation-induced sensitization of thymocytes to TRAIL-mediated apoptosis and expression of the apoptosis-inducing TRAIL receptors. However, with the use of several model systems, our subsequent experiments rule out the possibility that TRAIL plays a major role in antigen-induced deletion of thymocytes. In contrast to thymocytes, there is no up-regulation of TRAIL receptors in peripheral T cells on activation, which remain resistant to TRAIL. Thus, susceptibility to TRAIL-induced apoptosis is controlled differently by central and peripheral T cells.

Removing symbiotic Wolbachia bacteria specifically inhibits oogenesis in a parasitic wasp

Wolbachia are bacteria that live in the cells of various invertebrate species to which they cause a wide range of effects on physiology and reproduction. We investigated the effect of Wolbachia infection in the parasitic wasp, Asobara tabida Nees (Hymenoptera, Braconidae). In the 13 populations tested, all individuals proved to be infected by Wolbachia. The removal of Wolbachia by antibiotic treatment had a totally unexpected effect—aposymbiotic female wasps were completely incapable of producing mature oocytes and therefore could not reproduce. In contrast, oogenesis was not affected in treated Asobara citri, a closely related species that does not harbor Wolbachia. No difference between natural symbiotic and cured individuals was found for other adult traits including male fertility, locomotor activity, and size, indicating that the effect on oogenesis is highly specific. We argue that indirect effects of the treatments used in our study (antibiotic toxicity or production of toxic agents) are very unlikely to explain the sterility of females, and we present results showing a direct relationship between oocyte production and Wolbachia density in females. We conclude that Wolbachia is necessary for oogenesis in these A. tabida strains, and this association would seem to be the first example of a transition from facultative to obligatory symbiosis in arthropod–Wolbachia associations.

Tetrodotoxin-resistant Na+ currents and inflammatory hyperalgesia

Several mechanisms have been identified that may underlie inflammation-induced sensitization of high-threshold primary afferent neurons, including the modulation of voltage- and Ca2+-dependent ion channels and ion channels responsible for the production of generator potentials. One such mechanism that has recently received a lot of attention is the modulation of a tetrodotoxin (TTX)-resistant voltage-gated Na+ current. Evidence supporting a role for TTX-resistant Na+ currents in the sensitization of primary afferent neurons and inflammatory hyperalgesia is reviewed. Such evidence is derived from studies on the distribution of TTX-resistant Na+ currents among primary afferent neurons and other tissues of the body that suggest that these currents are expressed only in a subpopulation of primary afferent neurons that are likely to be involved in nociception. Data from studies on the biophysical properties of these currents suggest that they are ideally suited to mediate the repetitive discharge associated with prolonged membrane depolarizations. Data from studies on the effects of inflammatory mediators and antinociceptive agents on TTX-resistant Na+ currents suggest that modulation of these currents is an underlying mechanism of primary afferent neuron sensitization. In addition, the second-messenger pathways underlying inflammatory mediator-induced modulation of these currents appear to underlie inflammatory mediator-induced hyperalgesia. Finally, recent antisense studies have also yielded data supporting a role for TTX-resistant Na+ currents in inflammatory hyperalgesia. Although data from these studies are compelling, data presented at the Neurobiology of Pain colloquium raised a number of interesting questions regarding the role of TTX-resistant Na+ currents in inflammatory hyperalgesia; implications of three of these questions are discussed.

The spinal biology in humans and animals of pain states generated by persistent small afferent input

Behavioral models indicate that persistent small afferent input, as generated by tissue injury, results in a hyperalgesia at the site of injury and a tactile allodynia in areas adjacent to the injury site. Hyperalgesia reflects a sensitization of the peripheral terminal and a central facilitation evoked by the persistent small afferent input. The allodynia reflects a central sensitization. The spinal pharmacology of these pain states has been defined in the unanesthetized rat prepared with spinal catheters for injection and dialysis. After tissue injury, excitatory transmitters (e.g., glutamate and substance P) acting though N-methyl-d-aspartate (NMDA) and neurokinin 1 receptors initiate a cascade that evokes release of (i) NO, (ii) cyclooxygenase products, and (iii) activation of several kinases. Spinal dialysis show amino acid and prostanoid release after cutaneous injury. Spinal neurokinin 1, NMDA, and non-NMDA receptors enhance spinal prostaglandin E2 release. Spinal prostaglandins facilitate release of spinal amino acids and peptides. Activation by intrathecal injection of receptors on spinal C fiber terminals (μ,/∂ opiate, α2 adrenergic, neuropeptide Y) prevents release of primary afferent peptides and spinal amino acids and blocks acute and facilitated pain states. Conversely, consistent with their role in facilitated processing, NMDA, cyclooxygenase 2, and NO synthase inhibitors act to diminish only hyperalgesia. Importantly, spinal delivery of several of these agents diminishes human injury pain states. This efficacy emphasizes (i) the role of facilitated states in humans, (ii) shows the importance of spinal systems in human pain processing, and (iii) indicates that these preclinical mechanisms reflect processes that regulate the human pain experience.

Supraspinal contributions to hyperalgesia

Tissue injury is associated with sensitization of nociceptors and subsequent changes in the excitability of central (spinal) neurons, termed central sensitization. Nociceptor sensitization and central sensitization are considered to underlie, respectively, development of primary hyperalgesia and secondary hyperalgesia. Because central sensitization is considered to reflect plasticity at spinal synapses, the spinal cord has been the principal focus of studies of mechanisms of hyperalgesia. Not surprisingly, glutamate, acting at a spinal N-methyl-d-aspartate (NMDA) receptor, has been implicated in development of secondary hyperalgesia associated with somatic, neural, and visceral structures. Downstream of NMDA receptor activation, spinal nitric oxide (NO⋅), protein kinase C, and other mediators have been implicated in maintaining such hyperalgesia. Accumulating evidence, however, reveals a significant contribution of supraspinal influences to development and maintenance of hyperalgesia. Spinal cord transection prevents development of secondary, but not primary, mechanical and/or thermal hyperalgesia after topical mustard oil application, carrageenan inflammation, or nerve-root ligation. Similarly, inactivation of the rostral ventromedial medulla (RVM) attenuates hyperalgesia and central sensitization in several models of persistent pain. Inhibition of medullary NMDA receptors or NO⋅ generation attenuates somatic and visceral hyperalgesia. In support, topical mustard oil application or colonic inflammation increases expression of NO⋅ synthase in the RVM. These data suggest a prominent role for the RVM in mediating the sensitization of spinal neurons and development of secondary hyperalgesia. Results to date suggest that peripheral injury and persistent input engage spinobulbospinal mechanisms that may be the prepotent contributors to central sensitization and development of secondary hyperalgesia.