Studies on conditional gene expression in the brain.

This manuscript summarizes our recent attempts to regulate in vitro and in vivo the expression of genes encoding components and regulators of the postsynaptic machinery along with marker genes such as lacZ and GFP. In particular, we studied tTA-dependent regulation and utilized Cre in combination with reversible silencing by intron engineering of dominant negative alleles. We further present a "knockin" approach for on-site artificial regulation of chromosomal genes.

Neurological dysfunctions in mice expressing different levels of the Q/R site-unedited AMPAR subunit GluR-B.

We generated mouse mutants with targeted AMPA receptor (AMPAR) GluR-B subunit alleles, functionally expressed at different levels and deficient in Q/R-site editing. All mutant lines had increased AMPAR calcium permeabilities in pyramidal neurons, and one showed elevated macroscopic conductances of these channels. The AMPAR-mediated calcium influx induced NMDA-receptor-independent long-term potentiation (LTP) in hippocampal pyramidal cell connections. Calcium-triggered neuronal death was not observed, but mutants had mild to severe neurological dysfunctions, including epilepsy and deficits in dendritic architecture. The seizure-prone phenotype correlated with an increase in the macroscopic conductance, as independently revealed by the effect of a transgene for a Q/R-site-altered GluR-B subunit. Thus, changes in GluR-B gene expression and Q/R site editing can affect critical architectural and functional aspects of excitatory principal neurons.

Thalamic NMDA receptors modulate inflammation-produced hyperalgesia in the rat.

The effects of inhibition of thalamic NMDA receptor function and synthesis on thermal and mechanical hyperalgesia induced by hindlimb intraplantar injection of carrageenan in the rat were studied in the 'acute' phase (within 3-5 h) and the 'subacute' phase (24 h) after carrageenan administration. Blockade of NMDA receptors was produced by intrathalamic injection of D,2-amino-5-phosphonovaleric acid (D-APV) and NMDA receptor synthesis was decreased (or not) by pretreatment of rats with intrathalamic (hindlimb representation area) injections of antisense, sense or missense oligodeoxynucleotides (ODNs) directed against the NR1 subunit of the NMDA receptor complex. Treatment with D-APV, but not saline, in the contralateral (but not ipsilateral) thalamus significantly reduced both the acute thermal and mechanical hyperalgesia in the injected paw; these same rats demonstrated significantly less thermal and mechanical hyperalgesia in the sub-acute phase than rats that had received saline or D-APV in the ipsilateral thalamus. None of the treatments had any effect on withdrawal responses of the uninjected hindpaw. Rats pretreated with NR1 sense or missense ODNs developed both thermal and mechanical hyperalgesia that was equivalent in magnitude and duration to rats that received intrathalamic saline injections. In contrast, rats pretreated with NR1 antisense ODN did not develop either acute or subacute thermal hyperalgesia; they developed less mechanical hyperalgesia than saline, sense or missense ODN-treated rats. Antisense ODN-treated rats also displayed a decrease in the number of thalamic NMDA receptors as determined by receptor binding assay. These results suggest an involvement of thalamic NMDA receptors in the development and maintenance of hyperalgesia associated with neurogenic inflammation in a model of tonic pain.

Rapid construction in yeast of complex targeting vectors for gene manipulation in the mouse.

Targeting vectors for embryonic stem (ES) cells typically contain a mouse gene segment of >7 kb with the neo gene inserted for positive selection of the targeting event. More complex targeting vectors carry additional genetic elements (e.g. lacZ, loxP, point mutations). Here we use homologous recombination in yeast to construct targeting vectors for the incorporation of genetic elements (GEs) into mouse genes. The precise insertion of GEs into any position of a mouse gene segment cloned in an Escherichia coli/yeast shuttle vector is directed by short recombinogenic arms (RAs) flanking the GEs. In this way, complex targeting vectors can be engineered with considerable ease and speed, obviating extensive gene mapping in search for suitable restriction sites.

Modulation of spinal visceral nociceptive transmission by NMDA receptor activation in the rat.

1. Thirty-three neurons in the L6-Sl spinal cord of 30 adult male Sprague-Dawley rats were characterized for responses to colorectal distention (CRD, 20-80 mmHg, 20 s) and convergent cutaneous receptive fields in the presence and absence of N-methyl-D-aspartate (NMDA; 1 microM) or D-serine (1 microM) administered locally by pressure ejection. 2. NMDA ejected locally increased the resting (spontaneous) activity, responses to CRD, postdistention afterdischarges, encoding of visceral nociception, and the size of convergent cutaneous receptive fields of some neurons. Facilitation of responses to noxious intensities of CRD (> or = 40 mmHg) was apparent between 30 s and 4 min after drug ejection. The slope of stimulus-response functions to graded intensities of CRD was increased significantly by NMDA, although mean response threshold was not significantly altered after NMDA ejection. 3. Facilitatory effects of NMDA on responses to CRD and increases in size of convergent cutaneous receptive fields were blocked or reversed by administration of the NMDA receptor antagonist, 5-amino-2-phosphono-valeric acid. 4. D-serine, an agonist at the glycine modulatory site on the NMDA receptor complex, generally mimicked the effects of NMDA on neurons responsive to CRD. The effects of D-serine were blocked by the glycine site antagonist 7-chloro-kynurenic acid (7-CK). 7-CK also blocked NMDA-produced effects on responses to CRD and increases in size of cutaneous receptive fields. 5. No differences were found between spinal neurons with and without documented long ascending projections with respect to effects of NMDA or D-serine. 6. These findings demonstrate involvement of spinal NMDA receptors in mediating hyperexcitability of spinal neurons to visceral nociceptive input and suggest an important contribution of spinal NMDA receptors in visceral hyperalgesic syndromes.

N-methyl-D-aspartate receptor-mediated changes in thermal nociception: allosteric modulation at glycine and polyamine recognition sites.

The effects of allosteric modulators of the N-methyl-D-aspartic acid receptor ion-channel complex on the nociceptive tail-flick reflex were studied in awake rats. Intrathecal administration of D-serine (100 fmol-1 mumol) but not L-serine or glycine to the lumbar spinal cord produced a facilitation of the tail-flick reflex at doses > or = 1 pmol (maximum at 0.5-1 min). Intrathecal pretreatment with the glycine modulatory site antagonist 7-chlorokynurenic acid (3 pmol) blocked both D-serine-produced and N-methyl-D-aspartate-produced facilitation of the tail-flick reflex. D-serine-produced facilitation was also blocked by intrathecal pretreatment with a N-methyl-D-aspartate receptor ion-channel blocker, MK 801 (100 fmol), or with an alternate substrate for nitric oxide synthase, NG-nitro-L-arginine-methyl ester (100 nmol). Intrathecal administration of spermine (0.01 nmol-3 mumol) produced biphasic effects on tail-flick latency accompanied by mechanical hyperesthesia and vocalization at greater doses. Spermine-produced facilitation (maximum with 0.01 nmol to 1 nmol at 1 min) was blocked by intrathecal pretreatment with MK 801 (100 fmol), NG-nitro-L-arginine-methyl ester (100 nmol) or the polyamine modulatory site antagonist, arcaine (10 nmol). Spermine-produced inhibition (maximum with 300 nmol at 2 min) was blocked by intrathecal administration of MK 801 (1 nmol). Intrathecal administration of the N-methyl-D-aspartate receptor antagonist, D-2-amino-5-phosphonopentanoic acid (1 nmol), blocked inhibition and uncovered a facilitation produced by 1 mumol spermine. In addition, spermine produced multi-stage motor effects (immediate- and late-onset). Intrathecal pretreatment with MK 801 (1 nmol) blocked only the immediate-onset motor effects while the late-onset motor effects were selectively blocked by pretreatment with the kappa opioid receptor antagonist, nor-binaltorphamine (200 nmol). Taken together, these data suggest that D-serine and spermine facilitate nociceptive transmission by positive allosteric modulation of the N-methyl-D-aspartate receptor ion-channel. Furthermore, activation of the N-methyl-D-aspartate receptor is also necessary to elicit the immediate-onset motor effects and inhibition of the tail-flick reflex produced by greater doses of spermine. Because kappa opioid receptors appear to be involved, the spermine-produced late-onset motor effects may involve endogenous dynorphin release.

NMDA and quisqualate modulation of visceral nociception in the rat.

The effects of N-methyl-D-aspartic acid (NMDA; 100 fmol-1 nmol) or quisqualic acid (QA; 10 pmol-10 nmol) on visceromotor and pressor responses to noxious colorectal distention (CRD; 40 mmHg, 20 s duration, interstimulus interval: 4 min) were studied in awake rats. Lesser doses of NMDA (100 fmol-1 pmol) administered intrathecally (i.t.) to the lumbar spinal cord produced a dose-dependent facilitation of visceromotor as well as pressor responses to CRD (maximum with 1 pmol NMDA at 1 min). The greatest dose tested (1 nmol) attenuated these responses (maximum at 1 min) and also produced a caudally-directed biting and scratching behavior accompanied by vocalizations. NMDA did not produce any of the above effects when administered i.t. to the thoracic spinal cord. I.t. pretreatment with the NMDA receptor antagonist, D-2-amino-5-phosphonovaleric acid (D-APV; 1 pmol), which produced no change in baseline activity or control responses, blocked all NMDA-produced effects in a reversible manner. QA produced dose-dependent inhibitory effects on visceromotor as well as pressor responses to noxious CRD when given i.t. to the lumbar spinal cord but not on administration to the thoracic spinal cord. Three nmol QA produced maximum inhibition at 2 min after administration and also produced caudally-directed biting and scratching. All of the QA-produced effects were reversibly blocked by i.t. pretreatment with the non-NMDA receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX; 3 nmol), which produced no change in baseline activity or control responses. We also examined the effects of NMDA and QA on responses to graded intensities of CRD. One pmol NMDA selectively facilitated visceromotor responses to CRD at distention pressures of 40 and 80 mmHg but not at 20 mmHg. In contrast, 3 nmol QA inhibited visceromotor responses to CRD at all intensities tested. In summary, these data suggest that activation of NMDA and non-NMDA receptors in the spinal cord differentially modulates visceral nociceptive input. Spinal segmental NMDA receptor activation produces selective facilitation of visceral nociceptive processing at noxious intensities of stimulation and may thereby contribute to central mechanisms underlying visceral hyperalgesia.

Characterization of the role of spinal N-methyl-D-aspartate receptors in thermal nociception in the rat.

The effects of N-methyl-D-aspartate (100 amol-1 nmol) on the nociceptive tail-flick reflex were studied in awake rats. Lesser doses of N-methyl-D-aspartate (100 amol-10 pmol) administered intrathecally to the lumbar spinal cord produced a dose-dependent facilitation of the tail-flick reflex (maximum at 0.5-1 min). The greatest dose tested (1 nmol) inhibited the tail-flick reflex (maximum at 2-5 min) and produced a caudally directed scratching and biting behavior accompanied by vocalizations. Intrathecal pretreatment with the N-methyl-D-aspartate receptor antagonist, D-2-amino-5-phosphonovaleric acid (1 fmol-1 pmol), which produced no change in baseline tail-flick latency, blocked all N-methyl-D-aspartate produced effects in a dose-dependent manner (100 fmol D-2-amino-5-phosphonovaleric acid produced maximum blockage for about 40 min). The magnitude and duration of N-methyl-D-aspartate-produced biphasic effects on tail-flick latency were similar in awake and lightly pentobarbital-anesthetized rats, but caudally directed biting and scratching behavior was not produced in lightly anesthetized rats. Reversible spinalization at T8-T10 in lightly anesthetized rats (produced by cold-block) completely abolished inhibition of the tail-flick reflex produced by 1 nmol N-methyl-D-aspartate whereas facilitation produced by 10 pmol N-methyl-D-aspartate remained unchanged, indicating that N-methyl-D-aspartate-produced facilitation is a local, segmental effect and that N-methyl-D-aspartate-produced inhibition requires a supraspinal loop. To examine the nature of the supraspinal loop, potential contributions of descending noradrenergic and serotonergic systems were studied. Intrathecal pretreatment with 100 nmol phentolamine completely blocked N-methyl-D-aspartate-produced inhibition of the tail-flick reflex, while N-methyl-D-aspartate-produced facilitation and caudally directed biting and scratching behavior remained unchanged. Intrathecal pretreatment with 50 nmol methysergide reversed the inhibitory effect of 1 nmol N-methyl-D-aspartate, resulting in a potent and prolonged facilitation which could be blocked by D-2-amino-5-phosphonovaleric acid. (1 pmol). Intrathecal pretreatment with an alternate substrate for nitric oxide synthase, NG-nitro-L-arginine methyl ester (100 nmol), completely blocked N-methyl-D-aspartate-produced facilitation of the tail-flick reflex, whereas N-methyl-D-aspartate-produced inhibition and caudally directed biting and scratching behavior were unaffected.(ABSTRACT TRUNCATED AT 400 WORDS)