A. John MacLennan

An essential role for the H218/AGR16/Edg‐5/LPB2 sphingosine 1‐phosphate receptor in neuronal excitability

A wealth of indirect data suggest that the H218/AGR16/Edg‐5/LPB2 sphingosine 1‐phosphate (S1P) receptor plays important roles in development. In vitro, it activates several forms of development‐related signal transduction and regulates cellular proliferation, differentiation and survival. It is expressed during embryogenesis, and mutation of an H218‐like gene in zebrafish leads to profound defects in embryonic development. Nevertheless, the in vivo functions served by H218 signalling have not been directly investigated. We report here that mice in which the H218 gene has been disrupted are unexpectedly born with no apparent anatomical or physiological defects. In addition, no abnormalities were observed in general neurological development, peripheral axon growth or brain structure. However, between 3 and 7 weeks of age, H218–/– mice have seizures which are spontaneous, sporadic and occasionally lethal. Electroencephalographic abnormalities were identified both during and between the seizures. At a cellular level, whole‐cell patch‐clamp recordings revealed that the loss of H218 leads to a large increase in the excitability of neocortical pyramidal neurons. Therefore, H218 plays an essential, unanticipated and functionally important role in the proper development and/or mediation of neuronal excitability.

Chronic ethanol administration decreases brain-derived neurotrophic factor gene expression in the rat hippocampus.

We have previously demonstrated that chronic ethanol consumption decreases neurotrophic activity in hippocampal extracts, as assessed by a chick dorsal root ganglia bioassay, but has no effect on hippocampal NGF mRNA or NGF protein levels. We presently report that hippocampal mRNAs encoding neurotrophin-3 and basic fibroblast growth factor are also unaffected. However, in contrast, brain-derived neurotrophic factor mRNA is reliably decreased, thereby suggesting that ethanol-induced damage of the septohippocampal system may at least partially result from an ethanol-induced decrease in hippocampal brain-derived neurotrophic factor expression.

STAT3 phosphorylation in injured axons before sensory and motor neuron nuclei: Potential role for STAT3 as a retrograde signaling transcription factor

STAT3 is a latent transcription factor that is activated by plasma membrane growth factor receptor complexes. Conditional gene disruption data indicate that it contributes to the survival of cranial motor neurons after peripheral nerve lesion. In agreement, levels of activated STAT3 (Tyr705‐phosphorylated STAT3) have been shown to increase in the nuclei of adult cranial motor neurons during their regeneration after the same injury. The data presented here demonstrate that STAT3 is similarly but not identically affected in sciatic motor neurons after sciatic nerve injury. In addition, we find that sensory neuron nuclei also display an analogous increase in activated STAT3, thereby supporting a role for STAT3 in the survival and regeneration of these cells. Most interesting, the present data indicate that peripheral nerve lesion leads to a very rapid activation of STAT3 in axons at the lesion site. This response increases during the first 24 hours after injury and extends back to the motor and sensory neurons such that phospho‐STAT3–immunoreactive axons are first detected in the dorsal root ganglia and ventral spinal cord at the same postlesion time intervals at which the activated STAT3 is first detected in the neuronal nuclei. Together these data raise the possibility that axonal STAT3, activated at the injury site, acts as a retrograde signaling transcription factor, which promotes the survival and regeneration of both sensory and motor neurons. J. Comp. Neurol. 474:535–545, 2004.

The use of conditioned defensive burying to test the effects of pimozide on associative learning

Rats shocked by a wire-wrapped prod mounted on the wall of the experimental chamber buried the prod with available bedding material when they were tested 24 hr later. Injection of the neuroleptic, pimozide (1.0 mg/kg) before conditioning and again before testing disrupted this conditioned defensive burying; however, a concomitant reduction in general activity suggested that this deficit in conditioned burying may have reflected a general motor impairment instead of a learning deficit. The observation that rats conditioned under the influence of pimozide but tested 24 hr later while undrugged did not display deficits in conditioned burying confirmed this view. Thus, neuroleptics appear to disrupt learned behavior by interfering with the performance of conditioned responses rather than by disrupting associative learning per se.

Promotion of the development of enteric neurons and glia by neuropoietic cytokines: Interactions with neurotrophin-3

Neurotrophin-3 (NT-3) is known to promote enteric neuronal and glial development. Ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) were investigated to test the hypothesis that the development of subsets of enteric neurons and/or glia is also affected by a neuropoietic cytokine, by itself, or together with NT-3. Crest-derived cells, immunoselected from the fetal rat gut (E14) with antibodies to p75NTR, were found by RT-PCR and immunocytochemistry (after culture) to express both alpha (CNTER alpha) and beta components (gp130 and LIFR beta) of the tripartite CNTF receptor. In situ, myenteric ganglia below the esophagus were CNTFR alpha-immunoreactive by E16-E18. In vitro, CNTF and LIF induced in crest-derived cells nuclear translocation of STAT3 (signal transducer and activator of transcription 3), a concentration-dependent increase in expression of neuronal or glial markers, and a decrease in expression of the precursor marker, nestin. LIFR beta was expressed by more cells than CNTFR alpha; therefore, although the factors were equipotent, the maximal effect of LIF > CNTF. The cytokines and NT-3 were additive in promoting neuronal but not glial development. Specifically, the development of neurons expressing NADPH-diaphorase activity (an early marker found in inhibitory motor neurons) was promoted by CNTF and NT-3. These observations support the idea that a ligand for the tripartite CNTF receptor complex plays a role in ENS development.

The S1P2 sphingosine 1-phosphate receptor is essential for auditory and vestibular function.

Sphingosine 1-phosphate (S1P) is an endogenous growth factor with potent effects on many different cell types. Most of these effects are produced by activation of one or more of a family of G-protein coupled receptors. The S1P2 receptor can mediate S1P-induced proliferation, differentiation and survival in a wide variety of cells in culture. However, identifying essential in vivo functions for S1P2 has been hampered by its ubiquitous expression and the failure to detect any anatomical abnormalities in initial analyses of S1P2 knockout mice. We report here that all S1P2 knockout mice are profoundly deaf from postnatal day 22 and approximately half display a progressive loss of vestibular function with aging. Anatomically, both the auditory and vestibular systems appear to develop normally but then degrade. Morphological defects associated with hearing are first detected at 3 weeks postnatal as deformations of the organ of Corti/Nuels space. By one year of age structures within the scala media are dramatically altered. The S1P2 knockout mice also display a loss of otoconia consistent with the vestibular impairment. The present data are the first to indicate that S1P signaling plays critical roles, in vivo, in auditory and vestibular functions. The data further establish that the S1P signaling occurs through the S1P2 receptor and makes an essential contribution to the structural maintenance of these systems, raising the possibility that properly targeted enhancement of this signaling may prove to be clinically beneficial.

Conditioned analgesia in the rat

It has been suggested by recent studies that the analgesic reaction to electric shock can be conditioned. However, these studies either lacked shocked controls or used an indirect measure of analgesia (freezing). In the present investigation, each rat was exposed an equal number of times to two distinct environmental contexts. The rats were shocked in one context and reexposed to the same context before test, shocked in one context and reexposed to the nonshock context before test, or not shocked at all and reexposed to one of the two contexts. Immediately following reexposure, the pain reactivity of the rats was assessed by a hot plate (Experiment 1) and a tail-flick apparatus (Experiment 2). It was found that rats that were reexposed to the context in which they had been shocked were significantly more analgesic than rats in the other two groups (which did not differ). These results confirm that it is possible to condition shock-induced analgesia in the rat.

Effects of short- and long-term haloperidol administration and withdrawal on regional brain cholecystokinin and neurotensin concentrations in the rat

The effects of oral administration of the neuroleptic, haloperidol, on regional brain concentrations of cholecystokinin (CCK) and neurotensin were examined in the rat. Both short-term (3 weeks) and long-term (8 months) haloperidol administration increased the concentration of CCK in the substantia nigra. While short-term administration significantly increased the concentration of CCK in the ventral tegmental area and decreased the concentration of CCK in the cortex, including the medial prefrontal cortex, these effects were not observed following long-term drug administration. In contrast, long-term, but not short-term, haloperidol administration decreased the concentration of CCK in the olfactory tubercle. Withdrawal from long-term haloperidol did not alter CCK concentrations in any of the brain regions examined. Short-term haloperidol administration significantly increased the concentration of neurotensin in the caudate-putamen. Both short- and long-term administration increased the concentration of neurotensin in the nucleus accumbens, but only the increased following long-term administration reached statistical significance. Withdrawal from long-term haloperidol administration slightly decreased the concentrations of neurotensin in the caudate-putamen and nucleus accumbens. These results indicate that dopamine receptor blockade can affect both CCK- and neurotensin-containing neural systems. Furthermore, these two neuropeptides are affected differently depending upon the duration of haloperidol administration and withdrawal from this drug. The results raise the possibility that chronic administration of haloperidol may be toxic to some neurotensin-containing neurons in the basal ganglia.

Ciliary neurotrophic factor receptor alpha in spinal motoneurons is regulated by gonadal hormones.

Ciliary neurotrophic factor receptor α (CNTFRα) is the ligand-binding component of the CNTF receptor. CNTFRα expression is essential for the normal development of spinal motoneurons and is required for the development of a sex difference in motoneuron number in androgen-sensitive perineal motoneurons. We used immunocytochemistry to examine the expression and hormone regulation of CNTFRα protein in the spinal nucleus of the bulbocavernosus (SNB), dorsolateral nucleus and retrodorsolateral nucleus of the lower lumbar spinal cord of adult rats. CNTFRα immunoreactivity (CNTFRα-IR) was observed in the somata and dendrites of virtually all motoneurons. In all three motor pools, the intensity of motoneuron soma labeling was greatest among gonadally intact males and was reduced in females and gonadectomized males. The density of CNTFRα-IR in neuropil also tended to be highest in intact males. Short-term (2 d) testosterone propionate treatment reversed the decline in the density of soma labeling in the SNB of castrated males but did not reverse any other effects of castration. Long-term hormone treatment, achieved by implanting males with testosterone capsules at the time of gonadectomy, prevented the decline in soma labeling in all motor pools and partially prevented the decline in neuropil label caused by castration. We conclude that expression of CNTFRα protein is androgen-regulated in spinal motoneurons.

Ciliary neurotrophic factor receptor ?-immunoreactivity in the monkey central nervous system

Ciliary neurotrophic factor (CNTF) sustains the viability and phenotypic expression of a variety of neuronal populations in the central nervous system. Cranial and spinal motor neurons are particularly sensitive to the trophic effects of CNTF, and clinical trials are underway testing the potential therapeutic value of this trophic factor in patients with amyotrophic lateral sclerosis. Yet, the distribution of the alpha subunit of the receptor for ciliary neurotrophic factor (CNTFRα), which is essential for the trophic effects of CNTF to occur, is unknown in any primate species. Towards this end, the present study used a polyclonal antibody directed against CNTFRα to evaluate the distribution of CNTFRα‐immunoreactive (‐ir) cells within the brain and spinal cord of Cebus apella monkeys. CNTFRα‐ir was found exclusively within neurons. In the anterior horn of the spinal cord, virtually all motor neurons were darkly immunoreactive for CNTFRα. A similar pattern of CNTFRα‐ir was seen within all cranial motor nuclei with general somatic efferent function (III, IV, motor V, VI, VII, and XII cranial nerves). CNTFRα‐ir was also seen in other regions involved with motor function including the Purkinje cells of the cerebellum, the substantia nigra pars compacta, red nucleus, dorsal motor nucleus of X cranial nerve, and giant neurons of sensory motor neocortex. A few CNTFRα‐ir neurons were seen within the globus pallidus with concomitant terminal‐like staining within the subthalamic nucleus. Autonomic regions such as the mesencephalic nucleus of the trigeminal nerve and the interomedial lateral cell column of the thoracic spinal cord also contained CNTFRα‐ir neurons. Finally, the hippocampus displayed dense CNTFRα‐ir within the pyramidal cell layer of the hippocampal formation and the granule cell layer of the dentate gyrus. The dense expression of this CNTFRα protein within regions subserving motor, autonomic, and sensory functions suggests that CNTFRα supports many central nervous system regions with diverse functions. J. Comp. Neurol. 377:365–380, 1997.