[Economical body platform for neonatal rats stereotaxic surgery]. / Plataforma economica para cirugia estereotaxica en rata neonata.

INTRODUCTION: Usually, most commercial platforms that adapt to the stereotaxic apparatus in neonatal rats or small animals, to carry out surgery are very expensive. Moreover, the operator must have certain experience in its handling. DEVELOPMENT: The present work presents two platforms, one of them made in acrylic and the other of expanded polystyrene. These adapt perfectly to conventional stereotaxic apparatus, while operator does not require a great entrainment to carry out the surgical procedure. Histological slides of the prefrontal cortex, ventral hippocampus and basolateral amygdala from adult rats (postnatal day 70), staining with cresyl violet are shown. The neonatal lesions were made at postnatal day 7 with ibotenic acid applied in the prefrontal cortex, ventral hippocampus or basolateral amygdala. CONCLUSIONS: The present data suggest that it is possible to carry out lesions or to apply drugs in neonatal rats, by using an acrylic or expanded polystyrene adaptor for the stereotaxic apparatus. These have the advantage of being economic and having a simple design. Also, the type of anesthesia used in neonatal lesion rats, is discussed.

Plataforma económica para cirugía estereotáxica en rata neonata / Economical body platform for neonatal rats sterotaxic surgery

Introducción. Usualmente, los aditamentos o plataformas comerciales que se adaptan a los aparatos estereotáxicospara realizar cirugía en ratas neonatas o animales pequeños como el ratón son muy costosos. Además, el operador debe contar con una cierta experiencia en su manejo. Desarrollo. Se describen dos nuevas plataformas, una de acrílico y otra de poliestireno expandido. Ambas plataformas se adaptan perfectamente a un aparato estereotáxico convencional y el operadorno requiere gran experiencia para efectuar dicho procedimiento quirúrgico. Se muestran cortes histológicos donde se observan las lesiones realizadas al séptimo día de edad, con ácido iboténico, por vía estereotáxica, en la corteza prefrontal, en elhipocampo ventral o en la amígdala basolateral, teñidos con violeta de cresilo, de ratas adultas (70 días de edad). Conclusiones. Los resultados sugieren que es posible realizar lesiones o aplicar sustancias por vía estereotáxica a ratas neonatas utilizandoestos dos soportes de acrílico o poliestireno expandido, que son de diseño sencillo, de fácil construcción y económicos. Adicionalmente, se revisa el tipo de anestesia utilizada en ratas neonatas

Introduction. Usually, most of commercial platforms that adapt to the stereotaxic apparatus in neonatal rats orsmall animals, to carry out surgery are very expensive. Moreover, the operator must have certain experience in its handling. Development. The present work presents two platforms, one of them made in acrylic and the other of expanded polystyrene. These adapt perfectly to conventional stereotaxic apparatus, while operator does not require a great entrainment to carry outthe surgical procedure. Histological slides of the prefrontal cortex, ventral hippocampus and basolateral amygdala from adult rats (postnatal day 70), staining with cresyl violet are shown. The neonatal lesions were made at postnatal day 7 with ibotenic acid applied in the prefrontal cortex, ventral hippocampus or basolateral amygdala. Conclusions. The present data suggest that it is possible to carry out lesions or to apply drugs in neonatal rats, by using an acrylic or expanded polystyrene adaptor for the stereotaxic apparatus. These have the advantage of being economic and having a simple design. Also, the type ofanesthesia used in neonatal lesion rats, is discussed

Circadian clock function in isolated eyestalk tissue of crayfish.

Electrical mass response of crayfish photoreceptors (electroretinogram) was recorded continuously for up to seven days in isolated preparations that consisted of the retina and lamina ganglionaris. Electroretinogram amplitude varied in a circadian manner with a nocturnal acrophase and a period of 22-23 h in preparations kept in darkness. Acclimatization of animals to reversed light/dark cycles resulted in a phase reversal of the rhythm in vitro. The per (period) gene of Drosophila has been implicated in the genesis of rhythms in insects and in vertebrates. Immunocytochemical staining with an antibody against the PER gene product revealed immunoreactivity in the retinal photoreceptors, as well as in cell bodies in the lamina ganglionaris. Labelled axons run distally towards the photoreceptors and proximally to other areas of the lamina.

Seasonal rhythm of red pigment concentrating hormone in the crayfish.

The content of red pigment concentrating hormone (RPCH) in the eye-stalk of the crayfish Procambarus clarkii varies seasonally, with maximum values during the summer months and the lowest values in winter. The responsiveness of tegumentary chromatophores to synthetic RPCH varies concurrently.

Localization and release of 5-hydroxytryptamine in the crayfish eyestalk

The content and regional distribution of 5-hydroxytryptamine (5-HT) in the crayfish eyestalk was determined by high-performance liquid chromatography. Levels of the 5-HT precursors l-tryptophan (L-TRP) and 5-hydroxytryptophan (5-OH-TRP), and of three metabolites, 5-hydroxytryptophol (5-HTPH), N-acetylserotonin (NA-5-HT) and 5-hydroxy-indole-3-acetic acid (5-HIAA), were also determined. The total content of 5-HT in the eyestalk was 95.4+/-49.3 pg mg-1 wet mass (mean +/- s.d., N=55) while the specific content was 9.6+/-4.9 fmol microg-1 protein (mean +/- s.d. N=5). 5-HT was present in all four ganglia of the eyestalk. The highest proportion was found in the medulla terminalis (40.2 %) and the lowest in the retina lamina ganglionaris (9.9 %), which also had the lowest specific content. Conversely, the highest specific content of L-TRP was in the retina lamina ganglionaris. 5-HT biosynthesis and metabolism were explored in isolated eyestalks. The monoamine oxidase blocker pargyline, at concentrations between 0.8 and 10 mmol l-1, elicited a dose-dependent increase in 5-HT content. The biosynthesis of 5-HT in the crayfish eyestalk is suggested by the presence of its immediate precursor (5-OH-TRP) and by the suppression of 5-HT synthesis induced by m-hydroxybenzyl-hydrazine (m-HBH), a blocker of 5-OH-TRP decarboxylase. The presence of immunopositive cell bodies and axons was demonstrated using an anti-5-HT antiserum. 5-HT-like immunopositivity was detected in various regions of the eyestalk. Efferent immunopositive axons were also identified in the optic nerve, and these may have originated in the protocerebral lobe of the supraoesophageal ganglion. The branchings of these axons were profusely distributed in the neuropile of the medulla terminalis. A basal level release of 5-HT was detected in isolated eyestalks. The amount recovered was increased two-to threefold after blocking 5-HT uptake with fluoxetine (1 micromol l-1). Incubation of eyestalks in solutions containing a high K+ concentration (80 mmol l-1) released 5-HT. Electrical stimulation of the optic nerve released 5-HT as a function of the intensity of stimulation. Both the basal and evoked release were suppressed by lowering the Ca2+ concentration in the medium. These observations support a role for 5-HT as a neurotransmitter or neuromodulator in the crayfish eyestalk.

Regional distribution and immunocytological localization of red pigment concentrating hormone in the crayfish eyestalk.

A polyclonal antibody was raised against synthetic tyrosinated crustacean red pigment concentrating hormone (RPCH-Tyr) with the sequence Tyr-Leu-Asn-Phe-Ser-Pro-Gly-Trp-NH2 with a tryptophan amide at the carboxyl terminal end. Its specificity was tested in comparison with peptides of similar structure. It appears to recognize the three to five residues near the carboxyl terminal. Native RPCH in the crayfish eyestalk was determined by two methods: (a) immunoenzymatic assay (ELISA) using the aforementioned antibody; and (b) bioassay on segments of isolated crayfish tegumentary epithelium. The unitary content in whole eyestalks was 5.5 +/- 1.0 nmol for samples (n = 18) taken at night. The regional distribution of RPCH content in the eyestalk was determined. The greatest proportion (40%) was found in the sinus gland, and the lowest in the retina plus lamina ganglionaris (6%). The medulla interna, medulla externa, and medulla terminalis contained similar proportions (about 16% each). The highest specific content was in the sinus gland (65.0 vs 24.4 pmol/micrograms protein for the whole eyestalk). Immunopositive neurons were identified in the various regions of the eyestalk. In 22 preparations, an average of 7 cells were identified in the ventromedial rim of the medulla terminalis, sending axons to the sinus gland, after branching in the neuropil of the medulla terminalis. Dorsally, 2 cells were identified in the medulla interna and 4 large cells and 11 small cells were located in the medulla externa in close proximity to the lamina ganglionaris: none of these cells appeared to project to the sinus gland. Profuse immunopositive fibers were found in the lamina ganglionaris projecting distally toward the base of the retina. Immunopositive axons were also found in the optic nerve.

The circadian system of crustaceans.

Crustaceans exhibit a variety of overt circadian rhythms. Observations on intact animals suggest the existence of more than one circadian pacemaker in the nervous system. Ablation experiments so far have been inconclusive in pin-pointing the location of putative pacemakers. However, various structures, most notably the optic peduncle, have been shown to sustain circadian rhythmicity in vitro. Retinal sensitivity and neurosecretory activity display circadian rhythms in the isolated optic peduncle, but they are also responsive to synchronizing influences from other regions of the central nervous system, most notably the supraesophageal ganglion. A model based on a number of circadian pacemakers distributed in the central nervous system best fits the experimental results at present. Coupling of rhythmicity between independent circadian pacemakers is likely to occur, and a neuroendocrine stage of integration has been proposed for several rhythms. Various entraining agents have been identified, and more than one may play a part in the synchronization of a given rhythm.

Modulation of crayfish retinal sensitivity by 5-hydroxytryptamine.

The responsiveness of crayfish retinal photoreceptors to light was enhanced by exposure to 5-hydroxytryptamine (5-HT), either following injection into whole animals or following topical application to isolated eyestalks or retinas. The effect was measured as an increment in the amplitude of the receptor potential, and was dose-dependent in the range 10(-6)-10(-3) mol l-1 (injected as a 0.1 microliter dose in intact animals). It was more pronounced at low levels of illumination and was reversibly blocked by methysergide. The enhancement was a consequence of a dual effect: (a) retraction of the proximal pigment granules within the photoreceptors, with a corresponding increase in the light-admittance function of the retina; and (b) a direct effect, facilitating a membrane conductance increase which mediated the generation of the receptor potential. A set of axons in the lamina ganglionaris with a 5-HT-like immunoreactivity was found in the vicinity of the photoreceptor axons. 5-HT antagonists were capable of blocking the physiological retraction of pigment granules in photoreceptors at night, suggesting that 5-HT acts as a modulator during the nocturnal phase of the circadian cycle in the crayfish retina.

Range of modulation of light sensitivity by accessory pigments in the crayfish compound eye.

The compound eye of the crayfish during dark adaptation undergoes an enhancement of light sensitivity within a range of 3 log units. Only 1 log unit can be explained by the increase in responsiveness of the retinula cells. The rest can be accounted for by the migration of the proximal and distal accessory pigments. In isolated retinas, with the distal pigment paralysed in light-adapted position and the proximal pigment only partially responsive, the sensitivity enhancement in darkness is reduced in more than 1 log unit. By hormonally inducing the expansion of the distal pigment while the rest of the system remains dark-adapted, there is a shift of one log unit in the V-log 1 curve. In a crayfish mutant devoid of the two dark accessory pigments, the sensitivity enhancement in dark adaptation only covers one log unit.