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D W Pfaff

Publications and source records attributed to D W Pfaff.

At least 163 records · Page 9Linked to original sources
PubMed1993

Luteinizing hormone-releasing hormone gene expression in the bovine brain: anatomical localization and regulation by ovarian state.

Experiments were conducted to identify neurons in the bovine brain that express the LHRH gene and to determine whether LHRH mRNA levels are influenced by the ovaries. Two groups of postpubertal heifers were utilized: heifers killed during the mid-luteal phase of the estrous cycle (LUTEAL, n = 5) and heifers killed 14-16 wk following ovariectomy (OVX, n = 5). In situ hybridization was performed through use of a 32P-end-labeled deoxyoligonucleotide (59 mer) complementary to the human LHRH mRNA sequence. LHRH-expressing neurons were identified in the diagonal band of Broca, the preoptic area, and the anterior hypothalamus in a manner consistent with immunocytochemical localization. Reduced silver grains, proportional to LHRH mRNA content, were quantified (in pixels, 45x objective) with an image analysis system. Expected serum hormone concentration differences between endocrine states were confirmed by radioimmunoassay for progesterone (LUTEAL > OVX, p < 0.01) and for LH (OVX > LUTEAL, p < 0.01). Compared to the OVX group, LUTEAL heifers had 34% fewer LHRH-expressing neurons (p < 0.05); on the average, these neurons possessed 28% fewer pixels/cell (p < 0.01), indicating fewer copies of LHRH mRNA per cell. When the numbers of pixels in all labeled cells were totalled, LUTEAL animals had 57% fewer pixels (p < 0.05) than did the OVX females--probably reflecting a decrease in LHRH synthetic capacity in the LUTEAL animals. Therefore, during the mid-luteal phase of the bovine estrous cycle, ovarian steroid (i.e., luteal progesterone) suppression of LHRH release (as reflected by serum LH) is coincident with decreased LHRH mRNA in the brain.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Induction of FOS immunoreactivity in oxytocin neurons after sexual activity in female rats.

Several lines of evidence suggest that centrally released oxytocin (OT) may act in the hypothalamic ventromedial nucleus (VMN) to facilitate sexual behavior in female rats primed with estradiol and progesterone. The present experiment used a marker of neuronal activation, expression of the transcription factor FOS, to locate OT neurons activated during sexual behavior. Sexual behavior significantly increased the percentage of OT neurons in the hypothalamic paraventricular nucleus (PVN) expressing FOS in rats treated with estradiol and progesterone, compared to hormone-treated, nonmated controls. The OT neurons colabelled with FOS immunoreactivity were found in regions of the PVN, but not in the supraoptic nucleus. As reported by others, FOS expression also was induced in the preoptic area and the VMN. Taken together with other evidence, the results are consistent with the hypothesis that endogenous OT is involved in female sexual behavior, and suggest that the source of oxytocinergic innervation to the oxytocin receptors in the VMN may be neurons in the PVN. Interestingly, FOS expression in the VMN occurred in cells in the vicinity of OT fibers.

Animals↗
PubMed1993

Detection of messenger RNA and low-abundance heteronuclear RNA with single-stranded DNA probes produced by amplified primer extension labeling.

We describe a procedure for detection of low-abundance cellular RNAs by in situ hybridization histochemistry, using single-stranded DNA probes produced by amplified primer extension labeling with Taq polymerase. We have used this approach to detect a number of high- and low-abundance RNA species and have found it to be a simple and reproducible method of obtaining sensitive probes for in situ hybridization studies. For example, DNA probes generated by amplified primer extension labeling can detect low-abundance heteronuclear RNAs in individual neurons. Since this procedure does not involve recombinant DNA technology or microbiological facilities, it should prove useful to a wide variety of investigators studying the regulation of gene expression at the cellular level.

Animals↗
PubMed1993

Estrogen increases HIP-70/PLC-alpha messenger ribonucleic acid in the rat uterus and hypothalamus.

In the ventromedial hypothalamus (VMH) of female rats, estrogen induces a protein isoform, HIP-70, whose sequence is identical to a protein reported to be phosphoinositol-specific phospholipase-C-alpha (PLC-alpha). Since previous studies explored induction only at the protein level, we examined both the distribution of HIP-70/PLC-alpha mRNA in various tissues and the effects of estrogen on HIP-70/PLC-alpha mRNA. Using slot blot analysis, we found that HIP-70/PLC-alpha mRNA is most abundant in pituitary, uterus, and VMH of female rats compared with other brain regions and tissues. Since these are target tissues for estrogen action, we examined the effects of estrogen on the abundance of HIP-70/PLC-alpha mRNA in these areas. Levels of HIP-70/PLC-alpha mRNA increased greater than 3-fold in the uterus 18 h after estrogen treatment. HIP-70/PLC-alpha mRNA in the VMH also increased about 35% 3 h after estrogen treatment. In situ hybridization corroborated the induction in the ventrolateral ventromedial hypothalamus. No effect of estrogen was observed on pituitary PLC-alpha mRNA. These results indicate that estrogen does increase HIP-70/PLC-alpha mRNA levels in certain tissues. Since the induction of HIP-70/PLC-alpha mRNA in VMH is relatively modest compared to the much larger induction of the HIP-70 protein isoform, regulation of HIP-70/PLC-alpha may entail both pre- and posttranslational mechanisms. Because members of the PLC family catalyze the hydrolysis of phosphatidyl inositol, potentially activating several secondary mediators (intracellular Ca2+, protein kinase-C, and eicanosoids), this second messenger pathway may mediate some effects of estrogen.

Animals↗
PubMed1993

Enduring consequences of neonatal treatment with antisense oligodeoxynucleotides to estrogen receptor messenger ribonucleic acid on sexual differentiation of rat brain.

Sexual differentiation of the mammalian brain is regulated by steroids during a critical developmental period, particularly by estradiol, which is believed to be aromatized in brain from gonadally derived testosterone. To ascertain the importance of neuronal estrogen receptor expression during sexual differentiation, we infused a 15-mer oligodeoxynucleotide antisense to the region of the translation start codon of estrogen receptor messenger RNA (mRNA), into the hypothalamus of 3-day-old rat pups. Two separate control treatments consisted of either a scrambled nucleotide sequence oligodeoxynucleotide, which had little homology to known mRNAs, or vehicle. Female pups either received a lightly androgenizing dose of testosterone 6 h after oligo infusion or were not hormone treated. Infusion of antisense oligo to estrogen receptor mRNA protected against many of the androgenizing effects of testosterone. Androgenized females infused with antisense oligo were significantly more likely to exhibit female sexual behavior in adulthood after treatment with estrogen plus progesterone and remained sensitive to the induction of wheel-running behavior by estrogen treatment seen in normal females, whereas the control androgenized females did not. Normal females did not exhibit any effects of antisense oligo treatment on sexual or locomotor behavior, but antisense oligo-treated normal females showed a trend (P = 0.09) toward disrupted estrous cyclicity and behaved differently in tests of open field behavior compared to controls. After killing, brains were processed for histology. Morphometric analysis of the sexually dimorphic nucleus of the preoptic area demonstrated a significantly smaller volume in antisense oligo-infused androgenized females compared with vehicle and scrambled oligo-infused controls. The sexually dimorphic nucleus volume was smaller still in normal females infused with antisense oligo, consistent with estrogen receptor activation playing an active role in sexual differentiation of the female brain. These results demonstrate the effectiveness of antisense oligodeoxynucleotides in permanently altering a developmental process if administered during a critical period.

Animals↗
PubMed1993

RB and Cdc2 expression in brain: correlations with 3H-thymidine incorporation and neurogenesis.

Expression of the cell cycle regulatory proteins RB and p34cdc2 was examined in the adult rat brain, with special emphasis on proliferation and neuronal differentiation in the hippocampal formation and olfactory bulb. RB-like immunoreactivity (RB-IR) was detected throughout the brain, with particularly intense staining observed in hippocampal pyramidal cells, pyriform cortex, and cerebellar Purkinje cells. Intense RB-IR and cdc2-IR were also detected in proliferating neuronal precursor cells in the subgranular region of the dentate gyrus and in the subependymal region extending from the anterior lateral ventricle into the olfactory bulb. Many of these cells developed into neurons as assessed by the expression of neuron-specific enolase (NSE) and, in the hippocampal formation, the expression of Fos-IR following pentylenetetrazol-induced seizure activity. A good correlation was observed between the number of proliferating cells expressing intense nuclear RB-IR staining and the number of thymidine-labeled cells that had differentiated into functional hippocampal neurons. A substantial decrease in RB-IR during differentiation was also observed and occurred prior to the expression of NSE. The possibility that the loss of RB may be necessary for neuronal differentiation to proceed is discussed.

Animals↗
PubMed1992

Sex differences in the regulation of heat shock protein 70 kDa and 90 kDa in the rat ventromedial hypothalamus by estrogen.

The regulation of heat shock proteins 70 kDa (hsp 70) and 90 kDa (hsp90) by estradiol (EB) was examined in the ventromedial hypothalamus (VMH) of gonadectomized adult female and male rats with and without estradiol replacement. Estradiol significantly elevated hsp70 and hsp90 protein concentrations in EB-treated VMH of females, but not males, sacrificed at 12 h following steroid hormone compared with oil-vehicle controls. These findings confirm our previous observations that EB induces hsp70 and hsp90 in the female VMH16,17. The sex differences for the two proteins may occur by different mechanisms, since basal hsp70 levels were higher in males than females, whereas hsp90 levels were similar for the two sexes.

Animals↗
PubMed1992

Axial muscle EMG responses evoked by cutaneous flank nerves in the female rat: effects of spinal transection, steroid hormones, and anesthesia.

In the lateral longissimus muscle (LL) of ovariectomized, female rats anesthetized with low surgical doses of urethane (1.0 g/kg), cutaneous reflexes with similar EMG and response patterns could be elicited from CNS-intact rats and from rats 24 h after complete thoracic spinal cord transection. The probability of eliciting a response to contralateral cutaneous nerve stimulation alone is much lower in rats with complete spinal transections compared to CNS-intact rats. For both CNS-intact and spinal-transected rats, responses to ipsilateral cutaneous nerve stimulation had a shorter latency and required significantly less current on average than responses to contralateral stimulation. The respective currents for eliciting threshold responses to ipsi- and contralateral stimulation are less for CNS-intact than spinal-transected rats. For both CNS-intact and spinal-transected rats, responses to bilateral cutaneous nerve stimulation were inconsistent in the same animal from run to run. With the variability of response at this anesthetic level, no consistent effects of progesterone (acute, i.v.) or estrogen (acute, i.v. and pretreatment, s.c.) were observed in spinal-transected rats. Intravenous progesterone reduced early, unilateral responses in CNS-intact rats anesthetized with 1.0 g of urethane/kg. For both CNS-intact and spinal-transected rats, additional anesthesia during EMG recording produced a gradual decline in response magnitude which could be recovered with a modest increase in stimulus intensity. However, spinal-transected rats appear to require less anesthesia to reduce comparable responses. The results suggest that supraspinal input is especially effective for facilitating contralateral cutaneous reflexes in back muscles, whereas it contributes more equally with afferent input and segmental circuitry to the efficacy of ipsilateral cutaneous reflexes.

Anesthesia↗
PubMed1992

Alpha 1-adrenergic agonists act on the ventromedial hypothalamus to cause neuronal excitation and lordosis facilitation: electrophysiological and behavioral evidence.

To see if activation of central alpha 1-adrenergic receptors can cause facilitation of lordosis in rats, the behavioral effects of centrally administered alpha 1-agonists, methoxamine (MA) and phenylephrine (PhE), and related agents were studied. In ovariectomized rats treated with estrogen, infusion of MA, PhE, or a beta-agonist isoproterenol, into the lateral ventricle, or bilateral infusions of MA or PhE into the ventromedial hypothalamus (VMH) facilitated lordosis. Conversely, intra-VMH infusion of the alpha 1-antagonist prazosin (PZ) inhibited lordosis. Intra-VMH infusion of isoproterenol or an alpha 2-agonist clonidine, had no effect. Neither was the intra-VMH infusion of MA effective if: (i) the rats were not primed with estrogen; (ii) the tips of the cannulae were outside the VMH; or (iii) it was preceded by an intra-VMH infusion of the alpha 1b-antagonist, chloroethylclonidine (CEC). These results not only verify implications from recent studies that alpha 1-receptors in the hypothalamus are important for lordosis facilitation, but further show that the adrenergic facilitatory effect are: (i) mediated specifically by alpha 1b-subtype of the alpha 1-receptor, (ii) estrogen-dependent, and (iii) site-specific to VMH. To investigate neural mechanisms potentially underlying the lordosis-facilitating effect of alpha 1-activation, the actions of MA and PhE on the electrical activity of single neurons of the ventromedial nucleus of the hypothalamus (VMN) in vitro were studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic alpha-Agonists↗
PubMed1992

Immunocytochemical demonstration of neural cell adhesion molecule (NCAM) along the migration route of luteinizing hormone-releasing hormone (LHRH) neurons in mice.

Contact between the developing forebrain and the ingrowing central processes of the olfactory, vomeronasal and terminal nerves is preceded by a migration of neural cell adhesion molecule (NCAM)-immunoreactive cells from the epithelium of the olfactory pit and the formation of an NCAM-immunoreactive cellular aggregate in the mesenchyme between the olfactory pit and the forebrain. The axons of the olfactory, vomeronasal, and terminal nerves, also NCAM-immunoreactive, grow into the cellular aggregate, which as development proceeds, becomes continuous with the rostral tip of the forebrain. The lateral and more rostral part of the cellular aggregate receives the ingrowing axons of the olfactory nerves and becomes the olfactory nerve layer of the olfactory bulb. The medial, more caudal part receives the central processes of the vomeronasal and terminal nerves. The vomeronasal nerve ends in the accessory olfactory bulb. The central processes of the terminal nerve end in the medial forebrain. Luteinizing hormone-releasing hormone (LHRH)-immunoreactive neurons, like the vomeronasal and terminal nerves, originate from the medial part of the olfactory pit. These LHRH cells migrate into the brain along and within a scaffolding formed by the NCAM-immunoreactive axons of the vomeronasal and terminal nerves, and they are never seen independent of this NCAM scaffold as they cross the nasal lamina propria. The results suggest that: (1) NCAM is likely to be necessary for scaffold formation, and (2) the scaffold may be essential for the subsequent migration of LHRH neurons into the brain. Because they aggregate, migrating LHRH-immunoreactive neurons, on which we did not detect NCAM immunoreactivity, may interact via other cell adhesion molecules (CAM). Inasmuch as the interaction between the LHRH-immunoreactive neurons and the NCAM-immunoreactive scaffold is heterotypic, the possibility of a heterophilic (NCAM to other CAM) interaction is not ruled out. These findings focus our attention on the functional role of NCAM in this migratory system.

Animals↗
PubMed1992

Gene memory in neuroendocrine and behavioural systems.

Several examples of sex steroid hormone actions on rat brain and behaviour show that initial hormone exposures may be followed by enduring neuronal alterations, apparent long after the hormone itself has disappeared. Precedents from non-neuronal systems led to the concept of 'gene memory'. We are studying genomic structural alterations in rat hypothalamic neurons to account for these effects. The preproenkephalin gene is turned on by oestradiol in rat brain neurons in a tissue-specific and genetic sex-specific manner. Levels of preproenkephalin mRNA in the ventromedial hypothalamus correlate tightly with oestradiol-dependent reproductive behaviour. Our results indicate a tissue-specific pattern of DNA methylation in the enkephalin promoter. Putative binding sites for several transcription factors have been described in the preproenkephalin gene promoter; a role for some of these factors in regulating expression of the gene has been demonstrated.

Animals↗
PubMed1992

Reticulospinal and reticuloreticular pathways for activating the lumbar back muscles in the rat.

These experiments tested hypotheses about the logic of reticulospinal and reticuloreticular controls over deep back muscles by examining descending efferent and contralateral projections of the sites within the medullary reticular formation (MRF) that evoke EMG responses in lumbar axial muscles upon electrical stimulation. In the first series of experiments, retrograde tracers were deposited at gigantocellular reticular nucleus (Gi) sites that excited the back muscles and in the contralateral lumbar spinal cord. The medullary reticular formation contralateral to the Gi stimulation/deposition site was examined for the presence of single- and double-labeled cells from these injections. Tracer depositions into Gi produced labeled cells in the contralateral Gi and Parvocellular reticular nucleus (PCRt) whereas the lumbar injections retrogradely labeled cells only in the ventral MRF, indicating that separate populations of medullary reticular cells project to the opposite MRF and the lumbar cord. In the second series of experiments the precise relationships between the location of neurons retrogradely labeled from lumbar spinal cord depositions of the retrograde trace, Fluoro-Gold (FG) and effective stimulation tracks through the MRF were examined. The results indicate that the Gi sites that are most effective for activation of the back muscles are dorsal to the location of retrogradely labeled lumbar reticulospinal cells. To verify that cell bodies and not fibers of passage were stimulated, crystals of the excitatory amino acid agonist, N-methyl-D-aspartate (NMDA) were deposited at effective stimulation sites in the Gi. NMDA decreased the ability of electrical stimulation to activate back muscles at 5 min postdeposition, indicating a local interaction of NMDA with cell bodies at the stimulation site. In the third series of experiments, electrical thresholds for EMG activation along a track through the MRF were compared to cells retrogradely labeled from FG deposited into the cervical spinal cord. In some experiments, Fast Blue was also deposited into the contralateral lumbar cord. Neurons at low threshold points on the electrode track were labeled following cervical depositions, indicating a direct projection to the cervical spinal cord. The lumbar depositions, again, labeled cells in MRF areas that were ventral to the locations of effective stimulation sites, primarily on the opposite side of the medulla. In addition, the lumbar depositions back-filled cells in the same cervical segments to which the Gi neurons project. These results suggest that one efferent projection from effective stimulation sites for back muscle activation is onto propriospinal neurons in the cervical cord, which in turn project to lumbar cord levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Effects of estrogen and fimbria/fornix transection on p75NGFR and ChAT expression in the medial septum and diagonal band of Broca.

NGF receptor-expressing cells located in the basal forebrain have recently been shown to contain estrogen (E) receptors (Toran-Allerand and MacLusky. 1989. Soc. Neurosci. Abstr. 15: 954). In the present study, we have examined the effects of E-treatment on p75NGFR and choline acetyltransferase (ChAT) expression by neurons in the medial septum (MS) and the vertical (VDB) and horizontal (HDB) limbs of the diagonal band of Broca using immunocytochemical and in situ hybridization techniques. First, since E-treatment has been shown to affect neuronal survival and to stimulate synaptic reorganization and growth within various regions of the brain, we hypothesized that E-treatment might attenuate the loss of p75NGFR immunoreactivity (IR) which occurs in the MS and VDB following transection of the fimbria/fornix. Contrary to our hypothesis, E-treatment did not attenuate the effects of fimbria/fornix transection. In fact, E-treatment alone produced a significant decrease in the number of p75NGFR-IR cells detected in the MS. Subsequent experiments confirmed that chronic E-treatment produces a down-regulation of both p75NGFR-IR and p75NGFR mRNA in the MS and VDB. In the MS, estrogen appeared to affect a subpopulation of p75NGFR-expressing neurons which were also affected by fimbria/fornix transection since the effects of these two treatments were not additive. In addition, effects of E-treatment on p75NGFR-IR were sex-specific (observed in females but not in males) and were reversible in the MS after 2 weeks, but not after 4 weeks (allowing 2 weeks recovery), of E-treatment. A time-course analysis revealed that effects of E-treatment on p75NGFR-IR were not observed until after 16 days (MS) or 30 days (VDB) of E-treatment and were preceded by a significant and transient increase in ChAT expression in both the MS and VDB. The data are consistent with the possibility that continuous, long-term exposure to gonadal steroids may contribute to a loss of p75NGFR-expressing neurons with age. In addition, the data suggest that p75NGFR expression may play a role in regulating the functioning of specific basal forebrain cholinergic neurons. Different mechanisms by which E-treatment might influence ChAT and p75NGFR expression in brain are discussed.

Animals↗
PubMed1992

Mu-, delta-, and kappa-opioid receptor agonists selectively modulate sexual behaviors in the female rat: differential dependence on progesterone.

Previous studies suggested that opioid receptor agonists infused into the lateral ventricles can inhibit (through mu receptors) or facilitate (through delta receptors) the lordosis behavior of ovariectomized (OVX) rats treated with estrogen and a low dose of progesterone. The present study investigated the behavioral and hormonal specificity of those effects using more selective opioid receptor agonists. Sexually experienced OVX rats were implanted stereotaxically with guide cannulae aimed at the right lateral ventricle. One group of rats was treated with estradiol benzoate (EB, 10 micrograms) 48 hr and progesterone (P, 250 micrograms) 4 hr before testing, whereas the other group was treated with EB alone. Rats were infused with different doses of the selective mu-receptor agonist DAMGO, the selective delta-receptor agonist DPDPE, or the selective kappa-receptor agonist U50-488. The females were placed with a sexually vigorous male in a bilevel chamber (Mendelson and Gorzalka, 1987) for three tests of sexual behavior, beginning 15, 30, and 60 min after each infusion. DAMGO reduced lordosis quotients and magnitudes significantly in rats treated with EB and P, but not in rats treated with EB alone. In contrast, DPDPE and U50-488H increased lordosis quotients and magnitudes significantly in both steroid-treatment groups. Surprisingly, measures of proceptivity, rejection responses, and level changes were not affected significantly by mu or kappa agonists, although proceptivity and rejection responses were affected by DPDPE treatment. These results suggest that the effects of lateral ventricular infusions of opioid receptor agonists on the sexual behavior of female rats are relatively specific to lordosis behavior. Moreover, the facilitation of lordosis behavior by delta- or kappa-receptor agonists is independent of progesterone treatment, whereas the inhibitory effect of mu-receptor agonists on lordosis behavior may require the presence of progesterone.

Animals↗