Cells of origin of entorhinal cortical afferents to the hippocampus and fascia dentata of the rat.

The pathway from the entorhinal cortical region to the hippocampal formation has previously been shown to be comprised of two sub-systems, one of which projects predominantly to the ipsilateral fascia dentata and regio inferior of the hippocampus proper, and a second which projects bilaterally to regio superior. The goal of the present investigation was to determine if these two pathways might originate from different cell populations within the entorhinal area. The cells of origin of these entorhinal pathways were identified by retrograde labeling with horseradish peroxidase (HRP). Injections which labeled the entorhinal terminal fields in both the fascia dentata and regio superior resulted in the retrograde labeling of two populations of cells in the entorhinal area. Ipsilateral to the injection, HRP reaction product was found in the cells of layer II (predominantly stellate cells) and the cells of layer III (predominantly pyramidal cells). Contralateral to the injections, however, the reaction product was found almost exclusively in the cells of layer III. With selective injections of the entorhinal terminal field in regio superior, only the cells of layer III were labeled, but these were labeled bilaterally. Selective injection of the entorhinal terminal field in the fascia dentata, however, resulted in the labeling of cells of layer II, but not of layer III, and these cells of layer II were labeled almost exclusively ipsilaterally. A very small number of labeled cells in layer II were, however, found contralateral to the injection as well. No labeled cells were found either in the presubiculum or parasubiculum following injections of the hippocampal formation. These cell populations were found capable of retrograde transport of HRP, however, since cells in both presubiculum and parasubiculum were labeled following HRP injections into the contralateral entorhinal area. These results suggest that the projections to the fascia dentata originate from the cells of layer II, while the projections to regio superior originate from the cells of layer III of the entorhinal region proper. The very slight crossed projection from the entorhinal area to the contralateral area dentata probably originates from the small population of cells in layer II which are labeled following HRP injections in the contralateral area dentata.

Retrograde labeling of central nervous pathways with tritiated or Evans blue-labeled bovine serum albumin.

In an attempt to develop a technique for the retrograde tracing of neuronal pathways which does not depend on enzyme activity of horseradish peroxidase (HRP), we have examined the retrograde transport of bovine serum albumin (BSA) by neurons of the central nervous system. Following injections of either tritiated or Evans blue-labeled BSA into the hippocampal formation of the rat, labeled neurons were found in the ipsilateral entorhinal area in a pattern which was highly reminiscent of that obtained with HRP. The retrograde transport of BSA provides a method for the retrograde tracing of central nervous pathways which is completely independent of the HRP method, making it possible to use both substances for studies of collateral innervation, and can be used with any fixative, or no fixative at all.

Analysis of collateral projections with a double retrograde labeling technique.

A method is described whereby collateral innervation by a single cell type may be determined by double retrograde labeling with horseradish peroxidase (HRP) and tritiated bovine serum albumin ([(3)H]BSA). The projections from the entorhinal area to the hippocampal formation were analyzed as a model system, since layer III pyramidal cells project bilaterally to the hippocampus. Following HRP injections into the hippocampus of one side, and [(3)H]BSA injections into the opposite hippocampus, double labeled cells were found in layer III of the entorhinal cortex. These doubly labeled cells could best be viewed in plastic embedded material, whereas frozen sections were found to be inadequate for clearly distinguishing between silver grains and HRP reaction product.

Short-term estrogen replacement increases beta-preprotachykinin mRNA levels in uninjured dorsal root ganglion neurons, but not in axotomized neurons.

Dorsal root ganglion (DRG) neurons that mediate nociception express the high affinity NGF receptor (trkA) gene and the preprotachykinin (PPT) gene. NGF has been shown to regulate both of these DRG neuronal genes. Our laboratory has shown that these genes are also regulated by estrogen. Long-term daily estrogen replacement, in adult ovariectomized (OVX) rats, causes a coordinate decline in trkA and beta-PPT mRNA levels in lumbar DRG neurons, while short-term estrogen replacement increases trkA mRNA levels in uninjured as well as in axotomized lumbar DRG neurons. The purpose of the current study was to test the hypothesis that short-term estrogen replacement increases DRG beta-PPT mRNA levels in lumbar DRG neurons of OVX rats and that the increase is dependent on target-derived NGF. Sciatic nerve transection (SNT) was used to eliminate target-derived NGF in L4 and L5 DRGs in adult OVX rats. Seven days later, one-half of the SNT and one-half of the animals that had received sham sciatic nerve transactions (SHAM) received two daily injections of estradiol benzoate (EB). The remaining rats received two daily injections of vehicle alone. Quantitative in situ hybridization analyses of sections from L4 and L5 DRGs showed that two daily injections of EB significantly increased beta-PPT mRNA levels in DRGs of SHAM animals, but had no effect on beta-PPT mRNA levels in DRGs from SNT animals. These data coupled with our earlier observations of the effect of short-term estrogen replacement on DRG trkA mRNA levels, indicate that the regulation of DRG beta-PPT mRNA levels by estrogen requires target-derived NGF.

Estrogen regulates neurofilament gene expression in adult female rat dorsal root ganglion neurons.

Recently, adult female dorsal root ganglion (DRG) neurons were shown to express the estrogen receptor gene and to bind estrogen. This gene expression and binding is hormone dependent. Moreover, in a subpopulation of DRG neurons, the estrogen receptor is colocalized with high-affinity (trkA) and low-affinity (p75NGFR) receptors for nerve growth factor (NGF). In this NGF-responsive subpopulation of DRG neurons, estrogen regulates expression of the NGF receptor genes and may increase the sensitivity of these cells to the neurotrophin. The present study tested the hypothesis that neurofilament gene expression, which is regulated by NGF in these cells, is dependent on hormone status. In this study, ovariectomized (OVX) rats received either long-term physiological estrogen (conjugated estrogens; Premarin, Wyeth-Ayerst) replacement (low dose) or 10 times physiological levels (high dose). Quantitative in situ hybridization with an RNA probe for the 68-kDa neurofilament mRNA revealed a significant dose-dependent effect of Premarin on DRG neurofilament gene expression. In OVX animals receiving low-dose Premarin replacement therapy the mean steady-state 68-kDa mRNA level was as high as 4 times that of untreated OVX rats. High-dose therapy increased the mean 68-kDa neurofilament steady-state mRNA level to as much as six-fold that observed in untreated OVX animals. The estrogen-dependent upregulation of neurofilament gene expression appeared to occur in all DRG neurons, rather than in a subpopulation of those cells. These data suggest an important role for estrogen in the maintenance and function of primary sensory neurons. Whether estrogen directly regulates neurofilament gene expression or indirectly regulates it by increasing DRG neuronal sensitivity to neurotrophins or other growth factors remains to be determined.

Effects of short-term estrogen replacement on trkA mRNA levels in axotomized dorsal root ganglion neurons.

A population of adult dorsal root ganglion (DRG) neurons bind NGF with high affinity and express the trkA gene. In these cells, NGF regulates gene expression and function. Recently, a number of laboratories reported the presence of estrogen receptors in DRG neurons and profound effects of estrogen on DRG gene expression. Our laboratory, for example, has reported a significant and coordinate decrease in DRG trkA and beta-preprotachykinin (beta-PPT) mRNA levels following 90 days of daily estrogen injections to ovariectomized (OVX) rats. These data suggest, as has been suggested for medial septal cholinergic neurons, that estrogen may collaborate with NGF in the regulation of DRG neuronal gene expression and function. The current study examined further this potential collaboration in the DRG by determining the effect of short-term estrogen replacement in OVX rats on DRG trkA mRNA levels following sciatic nerve transection and the resulting removal of a vital source of NGF for those cells. In OVX rats, about 40% of lumbar DRG neurons contained trkA mRNA. Short-term estrogen replacement had no effect on the percentage of neurons containing trkA mRNA, but increased the mean trkA mRNA level in uninjured DRGs of OVX rats by 23%. Axotomy in OVX rats reduced the mean trkA mRNA level by 55% but did not significantly decrease the percentage of neurons containing the mRNA. Estrogen replacement, 7 days after axotomy, partially and significantly restored the mean trkA mRNA level. It was 49% greater than that of the untreated axotomized DRGs. It did not, however, significantly increase the percentage of DRG neurons containing trkA in axotomized DRGs. These observations show that short-term estrogen has an opposite effect on DRG neuronal trkA mRNA levels as compared to that of long-term estrogen demonstrated in our previous study. Moreover, the current data show that estrogen regulates trkA mRNA levels in the absence of target-derived NGF. These data suggest that estrogen may collaborate with NGF in the maintenance of normal adult DRG gene expression and function. Furthermore, these data suggest that loss of estrogen, such as that associated with menopause, may contribute to a decline in DRG neuronal function and an exacerbation of ongoing neuropathic processes.

Long-term estrogen replacement coordinately decreases trkA and beta-PPT mRNA levels in dorsal root ganglion neurons.

Estrogen status has profound effects on cutaneous sensitivity in adult female rats. The presence of alpha-estrogen receptor mRNA and protein in NGF-dependent, adult female rat dorsal root ganglion (DRG) neurons raises the possibility that estrogen modulates cutaneous sensation by acting directly on primary afferent neurons, perhaps by altering their sensitivity to NGF. The present study examined the effect of long-term (90 days) daily injections of an estrogen preparation, Premarin (Wyeth-Ayerst, Radnor, PA), to ovariectomized adult rats on lumbar DRG high-affinity NGF receptor, trkA, mRNA levels, and on beta-preprotachykinin (beta-PPT) mRNA levels, which have been shown to be regulated by NGF. Two doses were used in the experiments, the higher dose being 10 times that of the lower dose. Such injections had an effect opposite that reported for short-term, acute estrogen treatment on DRG trkA mRNA levels. The current data show that long-term daily estrogen treatment decreases trkA mRNA levels by 36%. After 90 days of estrogen treatment, no dose effect was evident. Moreover, as would be expected if beta-PPT gene expression is regulated by NGF through the trkA receptor, long-term estrogen treatment decreased DRG neuronal beta-PPT mRNA levels by about 30%. As with trkA, there was no dose effect evident after 90 days of estrogen treatment. These data suggest the possibility that estrogen modulates DRG neuropeptide gene expression and, perhaps, cutaneous sensitivity by regulating NGF receptor gene expression.

Development of nitric oxide synthase expression in the superficial dorsal horn of the rat spinal cord.

Development of nitric oxide synthase (NOS) expression in the superficial dorsal horn of the rat spinal cord was studied using NADPH diaphorase histochemistry. At birth, no positive staining was seen in the superficial laminae of the cord. A week later, a few small positive neurons and fibers were seen in presumptive lamina II. The adult pattern of NOS expression was evident by the end of the third postnatal week.