John A. O'Brien

Altered nociception, analgesia and aggression in mice lacking the receptor for substance P

The peptide neurotransmitter substance P modulates sensitivity to pain by activating the neurokinin-1 (NK-1) receptor, which is expressed by discrete populations of neurons throughout the central nervous system. Substance P is synthesized by small-diameter sensory ‘pain’ fibres, and release of the peptide into the dorsal horn of the spinal cord following intense peripheral stimulation promotes central hyperexcitability and increased sensitivity to pain. However, despite the availability of specific NK-1 antagonists, the function of substance P in the perception of pain remains unclear. Here we investigate the effect of disrupting the gene encoding the NK-1 receptor in mice. We found that the mutant mice were healthy and fertile, but the characteristic amplification (‘wind up’) and intensity coding of nociceptive reflexes was absent. Although substance P did not mediate the signalling of acute pain or hyperalgesia, it was essential for the full development of stress-induced analgesia and for an aggressive response to territorial challenge, demonstrating that the peptide plays an unexpected role in the adaptive response to stress.

Synchronization and maintenance of timekeeping in suprachiasmatic circadian clock cells by neuropeptidergic signaling.

Circadian timekeeping in mammals is driven by transcriptional/posttranslational feedback loops that are active within both peripheral tissues and the circadian pacemaker of the suprachiasmatic nuclei (SCN). Spontaneous synchronization of these molecular loops between SCN neurons is a primary requirement of its pacemaker role and distinguishes it from peripheral tissues, which require extrinsic, SCN-dependent cues to impose cellular synchrony. Vasoactive intestinal polypeptide (VIP) is an intrinsic SCN factor implicated in acute activation and electrical synchronization of SCN neurons and coordination of behavioral rhythms. Using real-time imaging of cellular circadian gene expression across entire SCN slice cultures, we show for the first time that the Vipr2 gene encoding the VPAC2 receptor for VIP is necessary both to maintain molecular timekeeping within individual SCN neurons and to synchronize molecular timekeeping between SCN neurons embedded within intact, organotypical circuits. Moreover, we demonstrate that both depolarization and a second SCN neuropeptide, gastrin-releasing peptide (GRP), can acutely enhance and synchronize molecular timekeeping in Vipr2-/- SCN neurons. Nevertheless, transiently activated and synchronized Vipr2-/- cells cannot sustain synchrony in the absence of VIP-ergic signaling. Hence, neuropeptidergic interneuronal signaling confers a canonical property upon the SCN: spontaneous synchronization of the intracellular molecular clockworks of individual neurons.

Analysis of Clock Proteins in Mouse SCN Demonstrates Phylogenetic Divergence of the Circadian Clockwork and Resetting Mechanisms

The circadian clock in the suprachiasmatic nuclei (SCN) is comprised of a cell-autonomous, autoregulatory transcriptional/translational feedback loop. Its molecular components include three period and two cryptochrome genes. We describe circadian patterns of expression of mPER2 and mPER3 in the mouse SCN that are synchronous to those for mPER1, mCRY1, and mCRY2. Coimmunoprecipitation experiments demonstrate in vivo associations of the SCN mPER proteins with each other and with the mCRY proteins, and of mCRY proteins with mTIM, but no mPER/mTIM interactions. Examination of the effects of weak and strong resetting light pulses on SCN clock proteins highlights a central role for mPER1 in photic entrainment, with no acute light effects on either the mCRY or mTIM proteins. These clock protein interactions and photic responses in mice are divergent from those described in Drosophila.

A Schwann cell mitogen accompanying regeneration of motor neurons

Motor neurons are the only adult mammalian neurons of the central nervous system to regenerate following injury. This ability is dependent on the environment of the peripheral nerve and an intrinsic capacity of motor neurons for regrowth. We report here the identification, using a technique known as messenger RNA differential display, of an extracellular signalling molecule, previously described as the pancreatic secreted protein Reg-2 (ref. 4), that is expressed solely in regenerating and developing rat motor and sensory neurons. Axon-stimulated Schwann cell proliferation is necessary for successful regeneration,, and we show that Reg-2 is a potent Schwann cell mitogen in vitro. In vivo, Reg-2 protein is transported along regrowing axons and inhibition of Reg-2 signalling significantly retards the regeneration of Reg-2-containing axons. During development, Reg-2 production by motor and sensory neurons is regulated by contact with peripheral targets. Strong candidates for peripheral factors regulating Reg-2 production are cytokines of the LIF/CNTF family, because Reg-2 is not expressed in developing motor or sensory neurons of mice carrying a targeted disruption of the LIF receptor gene, a common component of the receptor complexes for all of the LIF/CNTF family.

A diversity of paracrine signals sustains molecular circadian cycling in suprachiasmatic nucleus circuits

The suprachiasmatic nucleus (SCN) is the principal circadian pacemaker of mammals, coordinating daily rhythms of behavior and metabolism. Circadian timekeeping in SCN neurons revolves around transcriptional/posttranslational feedback loops, in which Period (Per) and Cryptochrome (Cry) genes are negatively regulated by their protein products. Recent studies have revealed, however, that these “core loops” also rely upon cytosolic and circuit-level properties for sustained oscillation. To characterize interneuronal signals responsible for robust pacemaking in SCN cells and circuits, we have developed a unique coculture technique using wild-type (WT) “graft” SCN to drive pacemaking (reported by PER2::LUCIFERASE bioluminescence) in “host” SCN deficient either in elements of neuropeptidergic signaling or in elements of the core feedback loop. We demonstrate that paracrine signaling is sufficient to restore cellular synchrony and amplitude of pacemaking in SCN circuits lacking vasoactive intestinal peptide (VIP). By using grafts with mutant circadian periods we show that pacemaking in the host SCN is specified by the genotype of the graft, confirming graft-derived factors as determinants of the host rhythm. By combining pharmacological with genetic manipulations, we show that a hierarchy of neuropeptidergic signals underpins this paracrine regulation, with a preeminent role for VIP augmented by contributions from arginine vasopressin (AVP) and gastrin-releasing peptide (GRP). Finally, we show that interneuronal signaling is sufficiently powerful to maintain circadian pacemaking in arrhythmic Cry-null SCN, deficient in essential elements of the transcriptional negative feedback loops. Thus, a hierarchy of paracrine neuropeptidergic signals determines cell- and circuit-level circadian pacemaking in the SCN.

The circadian cycle of mPER clock gene products in the suprachiasmatic nucleus of the Siberian hamster encodes both daily and seasonal time

The circadian clock in the hypothalamic suprachiasmatic nuclei (SCN) regulates the pattern of melatonin secretion from the pineal gland such that the duration of release reflects the length of the night. This seasonally specific endocrine cue mediates annual timing in photoperiodic mammals. The aim of this study was to investigate how changes in photoperiod influence the cyclic expression of recently identified clock gene products (mPER and mTIM) in the SCN of a highly seasonal mammal, the Siberian hamster (Phodopus sungorus). Immunocytochemical studies indicate that the abundance of both mPER1 and mPER2 (but not mTIM) in the SCN exhibits very pronounced, synchronous daily cycles, peaking approximately 12 h after lights‐on. These rhythms are circadian in nature as they continue approximately under free‐running conditions. Their circadian waveform is modulated by photoperiod such that the phase of peak mPER expression is prolonged under long photoperiods. mPER1 protein is also expressed in the pars tuberalis of Siberian hamsters. In hamsters adapted to long days, the expression of mPER1 is elevated at the start of the light phase. In contrast, there is no clear elevation in mPER1 levels in the pars tuberalis of hamsters held on short photoperiods. These results indicate that core elements of the circadian clockwork are sensitive to seasonal time, and that encoding and decoding of seasonal information may be mediated by the actions of these transcriptional modulators.

Expression of mCLOCK and other circadian clock-relevant proteins in the mouse suprachiasmatic nuclei.

Circadian timing in mammals is based upon the cell‐autonomous clockwork located in the suprachiasmatic nuclei (SCN) of the hypothalamus. It is thought to involve interlocked feedback loops in which periodic transcriptional drive to core clock genes is mediated by CLOCK/BMAL1 heterodimers. Negative‐feedback actions of the encoded proteins PER and CRY terminate this phase of the cycle. In lower species, rhythmic abundance of the mCLOCK homologue initiates the subsequent cycle. By contrast, it is proposed that the new circadian cycle in mammals is triggered by indirect, positive transcriptional actions leading to a subsequent surge in BMAL1. The aim of this study was to test predictions made by this model concerning the behaviour of the native clock factor mCLOCK in the mouse SCN. Using in situ hybridization, immunocytochemistry, Western blotting and immunoprecipitation, we demonstrate constitutive expression of mCLOCK as a nuclear antigen in the SCN. mCLOCK forms alternating, periodic associations with either mBMAL1 or the negative regulators mPER and mCRY. The results confirm predictions made by the ‘two‐loop’ model of the mouse clock, and further highlight the role of interlocked cycles of positive and negative transcriptional regulatory complexes at the heart of the circadian clockwork.

Modifications to the hand-held Gene Gun: improvements for in vitro biolistic transfection of organotypic neuronal tissue

Transfection and subsequent expression of DNA in living neuronal tissue is problematic and no technique has emerged that is completely non-damaging, efficient and reproducible. The Bio-Rad hand-held Gene Gun has overcome some of these problems by exploiting a biolistic method in which small gold particles carrying plasmid DNA are propelled into neurons whilst causing minimal detectable cell damage. In its current configuration, however, the Bio-Rad Gene Gun is optimised for transfecting cells in dispersed cultures, and therefore delivers particles superficially over a relatively wide area. Here we report modifications to the Bio-Rad Gene Gun that both enhance its accuracy by restricting its target area, and increase the depth penetration achieved by gold particles, thereby allowing smaller and deeper tissues to be transfected. These alterations make the modified Gene Gun more applicable for in vitro transfection of organotypic cultures and enhance its potential utility for in vivo gene delivery. Moreover, the modified configuration operates successfully at lower gas pressures, thereby reducing even further the degree of cell damage incurred during transfection.

Impaired IL-1β-induced neutrophil accumulation in tachykinin NK1 receptor knockout mice

Tachykinin NK1 receptors play an important role in the development of neurogenic inflammatory responses. We have used the murine air‐pouch model to investigate whether the neurogenic component of the cellular inflammatory response to interleukin‐1β (IL‐1β, 10 ng into the air‐pouch) is altered in NK1 receptor knockout mice compared to wild type controls. Air‐pouches were washed following a 4 h IL‐1β treatment, the wash collected and neutrophil number estimated using a Neubauer haemocytometer. The response to IL‐1β was significantly attenuated in NK1 receptor +/− (40% reduction) and −/− mice (62% reduction) compared to wild type controls (+/+), whilst the response to cytokine‐induced neutrophil chemoattractant (CINC, 0.3 μg) was unaffected. The response to substance P (7.5 nmol) was attenuated by approximately 50% in both NK1 receptor +/− and −/− mice compared to wild type controls. In conclusion NK1 receptors play a significant role in the cellular response to IL‐1β in a model of inflammation.

Diolistic labeling of neuronal cultures and intact tissue using a hand-held gene gun

Diolistic labeling is a highly efficient method for introducing dyes into cells using biolistic techniques. The use of lipophilic carbocyanine dyes, combined with particle-mediated biolistic delivery using a hand-held gene gun, allows non-toxic labeling of multiple cells in both living and fixed tissue. The technique is rapid (labeled cells can be visualized in minutes) and technically undemanding. Here, we provide a detailed protocol for diolistic labeling of cultured human embryonic kidney 293 cells and whole brain using a hand-held gene gun. There are four major steps: (i) coating gold microcarriers with one or more dyes; (ii) transferring the microcarriers into a cartridge to make a bullet; (iii) preparation of cells or intact tissue; and (iv) firing the microcarriers into cells or tissue. The method can be readily adapted to other cell types and tissues. This protocol can be completed in less than 1 h.