Temporal pattern of HRP spread from an iontophoretic deposit site and description of a new HRP‐gel implant method

Abstract

In the course of examining afferents to ventromedial hypothalamic (VMH) neurons using horseradish peroxidase (HRP), we needed to know how close to an iontophoretic deposit site neurons could be proved to be retrogradely labeled. In evaluating cells near but clearly outside HRP deposit sites visualized after a 24-hour survival period, for example, neurons which had been filled with HRP by somal or dendritic uptake could not be treated as retrogradely labeled and thus would not add to studies of intrahypothalamic connections. Rats were given standardized iontophoretic applications of HRP into VMH (continuous positive current 0.25 mu amp for 1 minute) and sacrificed after 5 or 15 minutes, 1, 4, 8, 12, or 24 hours in order to examine the pattern of HRP spread. The chromogen was tetramethylbenzidine. The volume of the application site visualized at 24 hours was less than 10% of maximum site size, which occurred at 1 hour. Since the cells located within the maximal spread boundary are candidates for nonretrograde labeling, HRP data on local connections obtained even from small iontophoretic deposits must be evaluated in the light of the demonstrated expansion and subsequent contraction of the application site. These results may also hold implications for the precision with which distant connections can be examined using the HRP retrograde method, as sites that appear discrete when visualized after 24-hour survival may have overlapped at shorter times post-iontophoresis. Incorporation of retrograde tracers into polyacrylamide gels provides an effective alternative to pressure injection or iontophoresis of aqueous tracer solutions. We describe a method for filling micropipettes with HRP-polyacrylamide gel. The pipettes are then implanted into brain sites to provide a confined pool of HRP. With postimplantation survival of 24 hour or longer, this method produces sites comparable in size to iontophoretic sites examined at 24 hours and results in improved retrograde labeling. Some results obtained with this method concerning the afferent connections of the dorsomedial hypothalamus are described.

The projection from medial geniculate to field AI in cat: organization in the isofrequency dimension

“…Further interpretation of this finding is complicated by the difficulty of determining the effective injection site, as opposed to the apparent injection site, in studies using retrograde tracer substances (for discussion, see, e.g., Mesulam, 1982;Fahrbach et al, 1984). Therefore, in cases with nearby injection sites, it cannot be decided whether the partial overlap of differently labeled MGv clusters is due to overlap of the effective injection sites in the cortex or to other factors, for example a certain scatter, i.e., a limited divergence, in the thalamocortical projection.…”

Microcapillary specifically designed for pressure microinjections of very low volumes

“…Although Fahrbach et al (1984) established that the sizes of iontophoretically administered horseradish peroxidase (HRP) injection sites tend to first increase and then decrease depending on the length of the postinjection period (the maximum size being achieved at 1 h after the injection, and then systematically decreased until 8 h postinjection) owing to diffusion and then clearing of the tracer, other authors (Staines et al, 1980) showed that this postinjection diffusion is very tracer dependent. For example, when HRP is conjugated with wheat germ agglutinin, the diffusion of the HRP molecule away from the site is impeded.…”

Differential Regulation of Luteinizing Hormone‐Releasing Hormone and Galanin Messenger Ribonucleic Acid Levels by Alpha1Adrenergic Agents in the Ovariectomized Rat

“…Saturating concentrations of each drug were suspended in a 9% polyacrylamide gel matrix designed to provide slow release over an extended period of time. We have successfully used these gels to provide chronic delivery of several GABAergic agents (28) and neuroanatomical tracers (31,32) into the brain. This drug-impregnated gel was mixed as previously described (31,32) and drawn into an inner cannula before it had time to solidify.…”

“…We have successfully used these gels to provide chronic delivery of several GABAergic agents (28) and neuroanatomical tracers (31,32) into the brain. This drug-impregnated gel was mixed as previously described (31,32) and drawn into an inner cannula before it had time to solidify. This inner cannula then fitted within the permanent guide cannula, and extended 1 mm beyond the tip of the guide cannula.…”