Dietary arachidonic acid and docosahexaenoic acid regulate liver fatty acid desaturase (FADS) alternative transcript expression in suckling piglets.

Molecular regulation of fatty acid desaturase (Fads) gene expression by dietary arachidonic acid (ARA) and docosahexaenoic acid (DHA) during early post-natal period, when the demand for long chain polyunsaturated fatty acids (LC-PUFA) is very high, has not been well defined. The objective of the current study was to determine regulation of liver Fads1, Fads2 and Fads3 classical (CS) and alternative transcripts (AT) expression by dietary ARA and DHA, within the physiological range present in human breast milk, in suckling piglets. Piglets were fed one of six milk replacer formula diets (formula-reared groups, FR) with varying ARA and DHA content from days 3-28 of age. The ARA/DHA levels of the six formula diets were as follows (% total fatty acid, FA/FA): (A1) 0.1/1.0; (A2) 0.53/1.0; (A3-D3) 0.69/1.0; (A4) 1.1/1.0; (D2) 0.67/0.62; and (D1) 0.66/0.33. The control maternal-reared (MR) group remained with the dam. Fads1 expression was not significantly different between FR and MR groups. Fads2 expression was down-regulated significantly in diets with 1:1 ratio of ARA:DHA, compared to MR. Fads2 AT1 expression was highly correlated to Fads2 expression. Fads3 AT7 was the only Fads3 transcript sensitive to dietary LC-PUFA intake and was up-regulated in the formula diets with lowest ARA and DHA contents compared to MR. Thus, the present study provides evidence that the proportion of dietary ARA:DHA is a significant determinant of Fads2 expression and LC-PUFA metabolism during the early postnatal period. Further, the data suggest that Fads3 AT7 may have functional significance when dietary supply of ARA and DHA are low during early development.

Pharmacology, distribution and development of muscarinic acetylcholine receptor subtypes in the optic tectum of Rana pipiens.

Visually evoked behaviors mediated by the frog optic tectum require cholinergic activity, but the receptor subtypes through which acetylcholine acts are not yet identified. Using quantitative autoradiography and scintillation spectrometry, we examined the binding of [3H]pirenzepine and [3H]AF-DX 384 in the laminated optic tectum of the frog. In mammalian systems, these substances bind excitatory (m1 and m3 subtypes) and inhibitory (m2 and m4 subtypes) muscarinic acetylcholine receptors, respectively. Pharmacological analyses, including the use of specific muscarinic toxins, confirmed the subtype selectivity of the radioligands in the frog brain. Binding sites for [3H]pirenzepine were distinct from those for [3H]AF-DX 384. In the adult tectum, [3H]pirenzepine demonstrated specific binding in tectal layers 5-9. [3H]Pirenzepine binding was also present in tadpoles as young as stage V, but all sampled stages of tadpole tectum had significantly less binding when compared to adults. Lesioning of the optic nerve had no effect on [3H]pirenzepine binding. Specific [3H]AF-DX 384 binding was found in all layers of the adult tectum. All sampled tadpole stages exhibited binding sites for [3H]AF-DX 384, but the densities of these sites were also significantly higher in adults than they were in developing stages. Short-term lesions of the optic nerve reduced [3H]AF-DX 384 binding in all tectal layers of the deafferented lobe when compared to the afferented one. Long-term lesions decreased [3H]AF-DX 384 sites in both lobes.These results indicate that multiple muscarinic acetylcholine receptor binding sites reside in the frog optic tectum at all stages of development, and their pharmacology resembles that of mammalian m1/m3, m2 and m4 subtypes. Our data indicate that few, if any, of these receptors are likely to be located on retinal ganglion cell terminals. Furthermore, the expression of inhibitory muscarinic subtypes seems to be regulated by different mechanisms than that for excitatory subtypes.

Distribution and development of nicotinic acetylcholine receptor subtypes in the optic tectum of Rana pipiens.

Acetylcholine allows the elicitation of visually evoked behaviors mediated by the frog optic tectum, but the mechanisms behind its effects are unknown. Although nicotinic acetylcholine receptors (nAChRs) exist in the tectum, their subtype has not been assessed. By using quantitative autoradiography, we examined the binding of [(3)H]cytisine and [(125)I]alpha-bungarotoxin in the laminated tectum. In mammalian systems, these radioligands bind with high affinity to alpha4 nAChR subunits and alpha7 nAChR subunits, respectively. [(3)H]Cytisine demonstrated high specific binding in adult frogs in retinorecipient layer 9, intermediate densities in layer 8, and low binding in layers 1-7 of the tectum. [(3)H]Cytisine binding was significantly higher in the tecta of adults than in those of tadpoles. Lesioning the optic nerve for 6 weeks decreased [(3)H]cytisine binding in layers 8/9 by 70+/-1%, whereas 6-month lesions decreased binding by 76+/-3%. Specific binding of [(125)I]alpha-bungarotoxin in adults was present only at intermediate levels in tectal layers 8 and 9, and undetectable in the deeper tectal layers. However, the nucleus isthmi, a midbrain structure reciprocally connected to the tectum, exhibited high levels of binding. There were no significant differences in tectal [(125)I]alpha-bungarotoxin binding between tadpoles and adults. Six-week lesions of the optic nerve decreased tectal [(125)I]alpha-bungarotoxin binding by 33+/-10%, but 6-month lesions had no effect. The pharmacokinetic characteristics of [(3)H]cytisine and [(125)I]alpha-bungarotoxin binding in the frog brain were similar to those demonstrated in several mammalian species. These results indicate that [(3)H]cytisine and [(125)I]alpha-bungarotoxin identify distinct nAChR subtypes in the tectum that likely contain non-alpha7 and alpha7 subunits, respectively. The majority of non-alpha7 receptors are likely associated with retinal ganglion cell terminals, whereas alpha7-containing receptors appear to have a different localization.

Activity-dependent regulation of substance P expression and topographic map maintenance by a cholinergic pathway.

We have assessed the role of activity in the adult frog visual system in modulating two aspects of neuronal plasticity: neurotransmitter expression and topographic map maintenance. Chronic treatment of one tectal lobe with the non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione decreased the percentage of substance P-like immunoreactive (SP-IR) tectal cells in the untreated lobe while disrupting topographic map formation in the treated one. Treatment with the NMDA receptor antagonist d-(-)-2-amino-5-phosphonovaleric acid (d-AP-5) disrupted the topographic map but had no affect on SP-IR cells. These results indicate that maintenance of the topographic map is dependent on direct input from the glutamatergic retinal ganglion cells, whereas substance P (SP) expression is being regulated by a pathway that relays activity from one tectal lobe to the other. Such a pathway is provided by the cholinergic nucleus isthmi, which is reciprocally connected to the ipsilateral tectum and sends a projection to the contralateral one. Mecamylamine and atropine, antagonists of nicotinic and muscarinic receptors, respectively, were used together to block all cholinergic activity or alone to block receptor subclass activity. All three treatments decreased SP expression and disrupted the topographic map in the treated tectal lobe. We conclude that both SP expression and topographic map maintenance in the adult optic tectum are activity-dependent processes. Although our results are consistent with the maintenance of the topographic map through an NMDA receptor-based mechanism, they suggest that SP expression is regulated by a cholinergic interaction that depends on retinal ganglion cell input only for its activation.