Biomedical subjects

D W Pfaff

Publications and source records attributed to D W Pfaff.

At least 217 records · Page 12Linked to original sources

PubMed1990

Quantitative in situ hybridization to measure single-cell changes in vasopressin and oxytocin mRNA levels after osmotic stimulation.

1. The measurement of cellular mRNA content by quantitative in situ hybridization is a valuable approach to the study of gene expression in brain since this tissue exhibits a high degree of phenotypic heterogeneity. 2. The cellular content of vasopressin and oxytocin mRNA in hypothalamo-neurohypophysial system neurons was altered by maintaining rats for 24 hr on 2% sodium chloride water. 3. Statistical and graphical techniques were then used to analyze cell by cell how mRNA levels were altered as a result of osmotic stimulation. We propose that the negative binomial probability distribution is a suitable model to describe how mRNA content varies across a defined cell population. For both measures of oxytocin and vasopressin mRNA levels, maximum-likelihood estimation indicated that this model adequately described empirical findings obtained from rats drinking tap water or salt water. 4. Both graphical and statistical analyses suggested how the defined neural system responds to osmotic stimulation: mRNA content was altered as a multiplicative function of "initial state." The utility and limitations of the quantitative approach are discussed.

Animals↗

PubMed1990

Hypothalamic effects on medullary reticular activation of deep back muscle EMG.

Medullary reticular stimulation can activate deep back muscle EMG in urethane-anesthetized female rats. Midbrain central gray stimulation can facilitate brainstem reticular control over deep back muscles. Since these deep back muscles lateral longissimus (LL) and medial longissimus (ML) execute the vertebral dorsiflexion of lordosis behavior, and since the motor control hierarchy sketched above parallels lordosis behavior circuitry, we tested the hypothesis that medial hypothalamic lesions (which, in behavioral experiments, decrease lordosis) can also reduce medullary reticular activation of deep back muscle EMG. Urethane-anesthetized rats were tested systematically for amplitude of lateral longissimus (LL) and medial longissimus (ML) EMG responses to electrical stimulus trains applied to the nucleus gigantocellularis (NGC) of the medullary reticular formation, before and after electrolytic lesions of the ventromedial hypothalamus (n = 18) or control sites (n = 30). Bilateral ventromedial hypothalamic lesions were able to greatly reduce EMG responses in LL and ML, often with a time course similar to previous lordosis behavioral results. Surprisingly, lesions at the anterior ventromedial nucleus pole were particularly effective, and may reflect importance of intraventromedial local neurons. Although, on the average, various control lesions were less effective, the ventromedial hypothalamic effect was not unique. For example, it was possible to see an EMG decrease following lesions of the dorsomedial thalamus. Nevertheless, EMG loss was not well correlated with changes in the cortical EEG, and thus does not appear to be a simple consequence of changes in "arousal." In conclusion, it appears that ventromedial hypothalamic neurons can affect medullary reticular control of back muscle EMG, but must share this role with other forebrain elements.

Animals↗

PubMed1990

Hypothalamic sites of progestin action on aggression and sexual behavior in female Syrian hamsters.

The effects of intracranial implants of the synthetic progestin, promegestone (R5020), on aggression and sexual behavior were examined in female Syrian hamsters. Ovariectomized female hamsters showed high levels of aggression and no lordosis towards stimulus male and female hamsters, both prior to and after estradiol benzoate treatment. Forty-six hr after estradiol treatment (10 micrograms SC), 10% crystalline promegestone was applied bilaterally (27 ga cannulae) to the ventromedial hypothalamus. When tested 5-7 hr later, these animals had a significant reduction in the rate of attacks towards females, with no lordosis responding to a male. In contrast, promegestone in the caudal anterior hypothalamus activated low levels of lordosis, yet these females maintained high levels of aggression towards stimulus females. Females receiving promegestone in the rostral anterior hypothalamus, and control females receiving intracranial cholesterol, maintained high levels of aggression and no lordosis. Estradiol treatment alone enhanced pelvic movements in response to experimenter-applied perineal stimulation in a majority of females (18/22 compared with 4/22 prior to estradiol injection), with intracranial treatments having no further effect. These results indicate that progestins act on aggression and sexual behavior in female Syrian hamsters at different hypothalamic loci.

Aggression↗

PubMed1990

Estradiol induction of proenkephalin messenger RNA in hypothalamus: dose-response and relation to reproductive behavior in the female rat.

Hormone effects on proenkephalin (PE) mRNA allow an opportunity to compare a brain region-specific molecular change with a quantifiable behavior. Slot blots were used to measure PE mRNA levels in the ventromedial hypothalamus (VMN) and preoptic area (POA) as a function of the dose of estrogen administered to ovariectomized rats. Every rat used had been characterized for the ability to display lordosis behavior. Estradiol treatment led to a monotonic dose-dependent increase in PE mRNA level in VMN, while only a small effect was observed in POA at the higher estradiol doses. Lordosis behavior, assessed manually and in mating tests, also increased monotonically with estradiol dose. The data indicate that an apparent 'threshold' level of PE mRNA in VMN coincided wit display of behavior, and suggest further that high levels of PE mRNA, alone, are not sufficient for lordosis. While the exact relationship of the eventual product, Met-enkephalin, to female reproductive behavior remains to be determined, the parallel changes in PE mRNA level and behavior encourage further analysis.

Animals↗

PubMed1990

Detection of vasopressin mRNA in the neurointermediate lobe of the rat pituitary.

A messenger ribonucleic acid (mRNA) homologous to the transcript that encodes vasopressin (VP) was detected in the neurointermediate lobe (NIL) of the rat pituitary. The abundance of this transcript is approximately 1/100th the amount detected in the hypothalamus. In rats drinking 2% NaCl-water for 0,2,4, or 10 days, or for 10 days and then tap water for 14 days, the levels of VP mRNA in the NIL were altered in a fashion that paralleled changes in the hypothalamus.

Animals↗

PubMed1990

Projections of ventromedial hypothalamic neurons to the midbrain central gray: an ultrastructural study.

Ventromedial hypothalamic projections to the midbrain central gray may be involved in the mediation of female reproductive behavior. In order to demonstrate and examine projections of the ventromedial nucleus in the midbrain central gray, in the rat, electrolytic lesions were placed in the ventromedial nucleus and the midbrain central gray was examined for ultrastructural signs of degeneration at various intervals, i.e. 27.5 h and two, four, six and eight days following the lesions. The fine structure of the midbrain central gray of unlesioned animals was also examined to characterize its normal morphology and to establish a baseline with which to compare the effects of the lesion. In unlesioned animals, the neuropil of midbrain central gray contained several synaptic types, with axodendritic synapses appearing to be the most predominant. Dendrites contained well-preserved microtubules. Synaptic endings contained many clear, round vesicles and some contained dense-cored vesicles as well. Neuropil synapses were both asymmetric and symmetric. Cell bodies were characterized by light cytoplasm and had asymmetric and symmetric synapses on their surface. Following electrolytic lesions in the ventromedial nucleus, various types of degenerative patterns were seen in the midbrain central gray, including electron-dense, flocculent, watery, and pinocytotic degeneration. Specific characteristics of degeneration included shrunken, dense axons and endings, clumped synaptic vesicles, abnormally large, dark mitochondria, membranous sacs of various sizes, swollen endings with reduced numbers of synaptic vesicles, and endings and processes containing large numbers of coated vesicles. Some of these signs were already evident at 27.5 h following the lesion. In addition, degenerating postsynaptic processes and cell bodies were seen in the midbrain central gray. At 27.5 h survival time, degenerating dendritic processes often appeared swollen, devoid of microtubules, and contained enlarged mitochondria. At longer survival times neuronal degeneration was observed in the midbrain central gray, characterized by electron-dense cell bodies and pycnotic nuclei. Both degenerating pre- and postsynaptic elements appeared to be engulfed by glial processes. Control lesions in non-hypothalamic regions which project to the midbrain central gray, i.e. nucleus gigantocellularis and pontine reticular formation and in a non-projecting region, i.e. parietal cortex, were performed.(ABSTRACT TRUNCATED AT 400 WORDS)

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PubMed1990

Transneuronal degeneration in the midbrain central gray following chemical lesions in the ventromedial nucleus: a qualitative and quantitative analysis.

In the preceding experiments with electrolytic lesions of the ventromedial nucleus of the hypothalamus, we showed pre- and postsynaptic degeneration in the midbrain central gray of the rat. The postsynaptic degeneration seen may indicate a transneuronal effect of the ventromedial nucleus on the midbrain central gray. Electrolytic lesions, however, destroy afferent endings and fibers in passage, so that the postsynaptic degeneration seen in the midbrain central gray may be due to retrograde degeneration of midbrain central gray afferents to the ventromedial nucleus or due to degeneration of fibers in passage. In order to distinguish among these possibilities, chemical, i.e. kainic acid and N-methyl aspartate, lesions were made in the ventromedial nucleus and the ultrastructure of the midbrain central gray and cerebral cortex was examined at various intervals following the lesions. Both of these excitotoxins have been shown to destroy neurons, sparing afferent terminals and fibers in passage. Animals receiving kainic acid lesions in the right ventromedial nucleus were allowed to survive for one week, and animals receiving N-methyl aspartate lesions in the right ventromedial nucleus were permitted to survive for four, eight, and 20 days. Midbrain central gray tissue of unlesioned animals served as a control for both kainic acid and N-methyl aspartate lesions. In addition, other control animals received injections of the same amount of N-methyl aspartate in the right parietal cortex and were permitted to survive for four and eight days. For each of the above injection and survival conditions, the left cortex and subdivisions of the midbrain central gray were removed and processed for electron microscopy. Animals receiving ventromedial hypothalamic lesions with both kainic acid and N-methyl aspartate showed signs of pre- and postsynaptic degeneration. A quantitative analysis (General Linear Model Procedure) of degeneration was performed on the cortex and midbrain central gray of animals receiving N-methyl aspartate lesions in the ventromedial nucleus and cortex, and several parameters were measured. Animals receiving ventromedial hypothalamic lesions and surviving for eight and 20 days show significantly higher ratios of degenerating presynaptic elements to total presynaptic elements, degenerating postsynaptic elements to total postsynaptic elements, and degenerating total elements to total elements, in the midbrain central gray than in the cortex. Furthermore, the ratio of degenerating postsynaptic elements to total postsynaptic elements is larger than the other ratios.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1990

Ipsilateral and contralateral effects on cutaneous reflexes in a back muscle of the female rat: modulation by steroids relevant for reproductive behavior.

1. Multiunit EMG recordings of cutaneous reflexes--evoked in the back extensor, lateral longissimus (LL), by bilateral stimulation of nerves to dorsal flank skin--were studied in ovariectomized female rats with and without estrogen pretreatment. 2. Poststimulus time (PST) histograms of data from rats with and without estrogen pretreatment show that the axial EMG response (10-30 ms) to ipsilateral (ipsi) flank skin nerve stimulation is significantly shorter in latency (1.4 ms) and 67% larger than the response to contralateral (contra) flank skin nerve stimulation recorded at the same site (n = 29 pairs). 3. When late EMG responses were also evoked at 50-120 ms in 37% of ipsi and 29% of contra cutaneous reflexes, the incidence of multiunit activity in the late peak was significantly greater in rats pretreated with silastics containing 100% estradiol (E) compared with 10% E or cholesterol controls. 4. When an ipsi cutaneous reflex in LL was conditioned by a stimulus to the contra flank skin nerve at a condition-test interval of 30 ms (C-T 30 ms), the average number of discharges in the early peak of the histogram was double that in the histogram obtained from the unconditioned ipsi reflex, independent of estrogen pretreatment. 5. In 12 out of 19 cases in which a contra cutaneous reflex was conditioned by a stimulus to the ipsi flank skin nerve (C-T 30 ms), the number of discharges in the early peak of the histogram was less than that in the early peak of the histogram derived from the unconditioned contra response, independent of estrogen pretreatment. 6. Intravenous injections of progesterone (P) or its metabolite 5 alpha-pregnane-3 alpha-ol-20-one (tetrahydraprogesterone, THP) decreased the magnitude of the early peak of the ipsi cutaneous reflex and the contra cutaneous reflex in LL, independent of estrogen pretreatment. At the same time, it did not reduce the magnitude of the early peak evoked by either ipsi or contra nerves after conditioning from the other side at C-T 30 ms. 7. As a consequence, the percentage facilitation of ipsi cutaneous reflexes by contra cutaneous conditioning was significantly increased after P or THP. This suggests that these steroids can selectively enhance behaviors involving bilateral inputs. 8. An unchanged axial motoneuron pool output to bilateral cutaneous inputs after P and THP, in spite of reduced motoneuron responses to cutaneous inputs from each side of the body, implies an increased gain in the reflex circuit.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1990

Ultrastructural characterization of prolactin-like immunoreactivity in rat medial basal hypothalamus.

Prolactin-like immunoreactivity has been reported in the medial basal hypothalamus at the light microscopic level, in hypophysectomized rats. Here, with preembedding immunocytochemistry at the electron microscopic level, we have observed prolactin-immunoreactive neurons and synapses in the hypothalamus. Reaction product was discovered in medial basal hypothalamic neurons, which had typical large nucleoli and received axosomatic synapses. In the cytoplasm, reaction product was distinctly granular. Immunoreactive neurons were usually surrounded by nonreactive cells. Reaction product was also seen in dendrites, some of which had spines. Some axons in the hypothalamus contained reaction product, usually surrounded by nonreactive axons, and immunopositive synapses were detected both in the hypothalamus and in the midbrain. In a small number of cases immunoreactive axons could be seen synapsing on immunoreactive dendrites.

Animals↗

PubMed1990

Chemical characterization of neuroendocrine targets for progesterone in the female rat brain and pituitary.

The secretory products of some of the cell types which respond directly to actions of progesterone in the female rat brain and pituitary were determined by combining immunocytochemistry with autoradiography following systemic administration of the synthetic progestin ligand [3H]-R5020. Four major findings are reported: (1) Approximately 90% of the tyrosine hydroxylase (TH)-immunoreactive neurons in the hypothalamic arcuate nucleus have progesterone receptors, while TH-immunoreactive neurons in other portions of the hypothalamus (e.g. the periventricular region and the zona incerta) do not. (2) Approximately 30% of the beta-endorphin neurons in the hypothalamus have progesterone receptors. (3) None of the luteinizing hormone-releasing hormone neurons examined have progesterone receptors. (4) Approximately 98% of the cells in the anterior pituitary that have progesterone receptors contain luteinizing hormone. Lactotrophs do not contain progesterone receptors. Many progestin targets in the brain remain to be characterized chemically. The implications for progesterone-inducible genes and neuroendocrine control systems are discussed.

Animals↗

PubMed1990

Terminations of LHRH-immunoreactive fibers in the subfornical organ of the opossum: an ultrastructural study.

Electron microscopic immunocytochemical approaches were used to analyze LHRH-containing elements in the subfornical organ of the opossum, a species in which this input to the subfornical organ is prominent. Not only were LHRH synaptic specializations easily demonstrated in the subfornical organ, forming axo-dendritic and axo-axonal contacts, but also LHRH-immunoreactive fibers contacted astrocytic end-feet on fenestrated capillaries and were found in the subependymal layer. LHRH-carrying elements in the subfornical organ may be important for relating reproductive functions to body fluid balance.

Animals↗

PubMed1990

Steroid hormone regulation of ribosomal RNA in rat hypothalamus: early detection using in situ hybridization and precursor-product ribosomal DNA probes.

In the female rat, behavioral and endocrine aspects of reproduction are controlled, in part, by the action of the steroid hormone estradiol on several regions of the brain, including the ventrolateral portion of the ventromedial hypothalamus (VL-VMN) and the arcuate nucleus of the hypothalamus (ARC). Quantitative assessment of the effects of estradiol on the regulation of ribosomal RNA in rat hypothalamus was accomplished in this study by tandem in situ hybridization experiments with 2 ribosomal DNA probes specific to the initial transcript (precursor) or mature, stable (product) rRNA. This novel approach allowed the regulation of RNA processing by steroid hormones to be analyzed in the individual neuron, a particularly important concern in heterogeneous tissue such as the brain. Estradiol was administered subcutaneously to ovariectomized rats for 15 min, 30 min, or 2 hr, or a discontinuous schedule of 2 hr on/7 hr off/2 hr on. Levels of precursor and product rRNA were measured in VL-VMN and ARC neurons using a computerized image-analysis system. Significant increases in the levels of precursor rRNA were observed only in the VL-VMN as early as 30 min after hormone exposure, with a doubling in the amount of precursor rRNA occurring at 2 hr. No changes in product rRNA were observed in either brain region at these early times. These data, in conjunction with our previous findings of increases in product rRNA after longer hormone exposure times, lead us to conclude that rRNA gene transcription is activated in rat hypothalamic neurons within 30 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Histamine excites arcuate neurons in vitro through H1 receptors.

The electrophysiological effects of histamine on neurons of the hypothalamic arcuate nucleus of the female rat were tested with extracellular single unit recordings in an in vitro slice preparation. Histamine increased the spontaneous neuronal firing rate in 63% of the arcuate cells tested. An inhibitory response to histamine was seen in only one of the 117 neurons tested. The excitatory response to histamine showed dose dependency and was stable during synaptic blockade (by high magnesium and low calcium concentrations) and across a temperature range of 29-37 degrees C. Administration of histaminergic type 1 (pyrilamine and chlorpheniramine) and type 2 (cimetidine) receptor blockers revealed that the excitatory responses to histamine were mediated by type 1 receptors. The same neurons were also tested for responses to norepinephrine, serotonin, acetylcholine and substance P. A significant correlation was found between responses to histamine and substance P: all units excited by substance P were also excited by histamine. This subclass of histamine-responsive arcuate neurons may play a role in the regulation of the anterior pituitary, since histamine and substance P have similar effects on LH and prolactin secretion.

Action Potentials↗

PubMed1989

Intrinsic synapses in the ventromedial nucleus of the hypothalamus: an ultrastructural study.

To test the ideas that neurons intrinsic to a hypothalamic region might participate in steroid hormone effects and that intrinsic synapses might have unusual morphological features, we deafferented the ventromedial nucleus of the hypothalamus (VMH) in ovariectomized female rats with a Halasz knife. Some rats were given estrogen; others were endocrine controls. The main result of this study was that a large number of synapses in VMH survived the circumscribing cut. In fact, they comprised between one-half and two-thirds the number counted in unoperated controls. These were apparently from neuron cell bodies intrinsic to the medial basal hypothalamic island. Their morphology was not clearly different from that expected from unoperated controls or from other brain regions. A significant estrogen effect, an increase in the number of axodendritic synapses per unit area as seen in animals without surgical transections, was not present in the deafferented groups. This suggests that the hormone effect is due to synapses on VMH neurons from cell groups outside the medial basal hypothalamic island. If functional effects of estrogen mediated through VMH do not rely on intrinsic neurons, they must use estrogen-concentrating neurons that project to other brain regions.

Animals↗

PubMed1989

Origin of luteinizing hormone-releasing hormone neurons.

Neurons expressing luteinizing hormone-releasing hormone (LHRH), found in the septal-preoptic nuclei and hypothalamus, control the release of gonadotropic hormones from the anterior pituitary gland and facilitate reproductive behaviour. LHRH-expressing neurons are also found in the nervus terminalis, a cranial nerve that is a part of the accessory olfactory system and which projects directly from the nose to the septal-preoptic nuclei in the brain. During development, LHRH-immunoreactivity is detected in the peripheral parts of the nervus terminalis before it is found in the brain. Using a combination of LHRH immunocytochemistry and tritiated thymidine autoradiography in fetal mice, we show that LHRH neurons originate in the medial olfactory placode of the developing nose, migrate across the nasal septum and enter the forebrain with the nervus terminalis, arching into the septal-preoptic area and hypothalamus. Clinically, this migratory route for LHRH-expressing neurons could explain the deficiency of gonadotropins seen in 'Kallmann's syndrome' (hypogonadotropic hypogonadism with anosmia).

Animals↗

PubMed1989

Steroid hormone effects on picrotoxin-induced seizures in female and male rats.

Picrotoxin (1 mg/kg, i.p.), evoked a single generalized seizure in 75% of ovariectomized rats. Pretreatment of matched pairs with silastic implants containing 100% estradiol had an anticonvulsant effect; it protected all rats against such seizures. Implants containing 10% estradiol in cholesterol were less effective in protecting against picrotoxin-induced seizures. With 2 mg/kg picrotoxin, 85% of the seizure-affected ovariectomized controls had multiple seizures. The incidence of seizures and the ratio of single to multiple seizures induced by the higher dose of picrotoxin were unaffected by estradiol silastic implants, intraperitoneal injections of progesterone (0.5 mg, 4-5 h before convulsant) or the combination of both hormones. At the 2 mg/kg dose, 8/8 intact males had no seizures while all paired ovariectomized females had seizures. By contrast, the incidence of seizures in pairs of gonadectomized males and females did not differ. Testosterone treatment improved the ratio of single to multiple seizures in males but not in females. Males had statistically fewer multiple seizures than did females after testosterone treatment. The distribution of latencies to a single seizure is statistically different from the distribution of latencies to the first of multiple seizures irrespective of dose, sex and hormone treatment. This suggests that the population of rats responding with a single seizure at the higher dose of picrotoxin have a higher threshold for acquiring multiple seizures and that testosterone predisposes males but not females to this population.

Animals↗

PubMed1989

GABA[A] receptor level changes in female hamster forebrain following in vivo estrogen progesterone and benzodiazepine treatment: a quantitative autoradiography analysis.

The levels of gamma amino butyric acid (GABAA) receptors (i.e. 3H-Muscimol binding sites) were determined by quantitative neurotransmitter receptors autoradiography in ovariectomized (OVX), OVX-estradiolo (E), OVX-progesterone (P), OVX-E + P, diazepam (DZ) and DZ + Ro15-1788 treated female hamsters. The various hormonal treatments altered 3H muscimol binding in many brain areas, whereas DZ and DZ + Ro15-1788 had little influence on GABAA receptor levels at the onset of the blocked aggressive behavioral activity. For example, E, P and E + P all significantly increased 3H muscimol binding in medial preoptic area, ventromedial hypothalamic nuclei, and vertical diagonal bandmedial septal nucleus, whereas P treatment increased binding in the caudate-putamen and decreased it in reuniens nucleus of the thalamus. E and E + P treatments increased 3H muscimol binding in the corticomedial amygdala nucleus and hippocampus. Diazepam treatment decreased the GABAA receptor binding in the caudate-putamen and basolateral amygdala, while having no effect in the other brain regions where hormone treatment was effective. In vitro incubation of brain sections with micromolar concentrations of E or P did not change muscimol binding. These results suggest that ovarian steroids, and benzodiazepines to a lesser extent, modulate 3H muscimol binding but do so in different brain regions. The hormone effects on 3H muscimol binding in the critical reproductive centres at a time period that coincides with the onset of its behavioral effect is consistent with a genomic controlled activity.

Animals↗

PubMed1989

Preprotachykinin gene expression in the mediobasal hypothalamus of estrogen-treated and ovariectomized control rats.

We have determined the localization of preprotachykinin (PPT) mRNA-containing neurons in the mediobasal hypothalamus of the rat. PPT mRNA-containing neurons are present in the ventromedial nucleus (with a concentration in the ventrolateral aspect (VL-VM], the dorsomedial nucleus, the lateral hypothalamus and the arcuate nucleus. This distribution is consistent with the findings of immunocytochemical studies of substance P-immunoreactive neurons in the hypothalamus. We have also examined whether PPT gene expression is regulated by estrogen in the VL-VM by comparing the levels of PPT mRNA in the VL-VM of ovariectomized rats and ovariectomized, estrogen-replaced rats. Both in situ hybridization and slot blot hybridization analysis revealed no changes in PPT mRNA content in the VL-VM following estrogen treatment. These results suggest that estrogen does not regulate lordosis behavior by affecting PPT gene expression in VL-VM neurons.

Animals↗