Identification of medial preoptic neurons that concentrate estradiol and project to the midbrain in the rat

Fluorescent dye retrograde tracing was combined with steroid hormone autoradiography to study the midbrain projections of the estrogen-concentrating neurons in the preoptic region of the rat brain. Microinjections of the dyes DAPI, true blue, or a mixture of DAPI and primuline were made into the ventral tegmental area and into the midbrain central gray of ovariectomized, adrenalectomized 2-3-month-old female rats; 3 or 4 days later these animals were injected with [3H]estradiol; the brains were then processed for autoradiography. After exposures of from 3 to 12 months, the autoradiograms were developed and examined for reduced silver grains under cell nuclei (indicating binding of [3H]estradiol) and retrogradely transported fluorescent dye in the cytoplasm (indicating an efferent projection to the midbrain). Numerous [3H]estradiol-concentrating neurons in the medial preoptic region were found to send their axons to the medial midbrain. The largest numbers of estrogen target neurons that were afferent to the ventral tegmental area and to the midbrain central gray were found in the medial preoptic nucleus, in the surrounding medial preoptic area, and in the ventral bed nucleus of the stria terminalis. Double-labeled neurons were also identified in the preoptic suprachiasmatic area, in the lateral preoptic area, and in the rostral anterior hypothalamic area. Thus, a subset of the gonadal steroid target cells of the preoptic region have long projections to the medial midbrain, and a subset of the medial preoptic neurons that project to the ventral tegmental area and to the midbrain central gray concentrate estrogen. Behaviors (for example, maternal behavior, male copulatory behavior, and wheel-running) that are regulated by estrogen action in the medial preoptic region may be controlled by the direct estrogen-sensitive pathway to the medial midbrain revealed in this study.     

Estradiol-concentrating forebrain and midbrain neurons project directly to the medulla

The location and number of estradiol (E2)-concentrating neurons afferent to the dorsal medulla were determined by combining retrograde fluorescent tract tracing with steroid hormone autoradiography. Injections of Fluro-Gold were made into the medulla of 80 day old, ovariectomized, and adrenalectomized female rats. After 7 days survival to allow for retrograde transport, females were injected with [3H]estradiol; they were then perfused and their brains processed for steroid hormone autoradiography. Following a 4-12 month exposure period, autoradiograms were developed and microscopically analyzed for the presence of E2-concentrating neurons that project to the medulla. Numerous E2-concentrating neurons were identified that send axons directly to the medulla; the majority were found in the bed nucleus of the stria terminalis, paraventricular nucleus of the hypothalamus, central nucleus of the amygdala, and the central gray. Of the E2-concentrating neurons in the bed nucleus of the stria terminalis, 12.7% also projected to the medulla. E2-concentrating neurons that sent axons to the medulla were also identified in and ventromedial to the lateral parvicellular subdivision in the caudal half of the paraventricular nucleus of the hypothalamus (69.4%). Over one-third of the E2-concentrating neurons found in the central nucleus of the amygdala coursed to the medulla. The central gray was the only mesencephalic brain region that contained E2-concentrating neurons that projected to the medulla (41.9%). The medulla-bound E2-concentrating forebrain and midbrain neurons identified in the present study may influence autonomic tone via direct projections.     

Osmosensitive neurons in the rat's dorsal midbrain.

In experiment 1, multiple unit recordings were taken simultaneously from lateral preoptic and dorsal midbrain areas during a series of intracarotid hypertonic and isotonic NaCl injections. Subjects were 15 hooded rats (11 males and 4 ovariectomized females) under urethane anesthesia. Results showed that the neuronal reactions to a series of hypertonic NaCl injections (0.30 M, 0.45 M, 0.60 M and 0.75 M) were at least as strong in the dorsal midbrain as in the lateral preoptic area. Strength of neuronal reaction correlated with osmolarity of the NaCl solution injected. Control isotonic NaCl injections were ineffective, and the (monitored) force of injection was found not to affect the results. In experiment 2 with 15 hooded rats (9 males and 6 ovariectomized females), and two male Wistar rats under urethane anesthesia, recording from dorsal midbrain units were made during intracarotid injections of hypertonic and isotonic NaCl solutions. In addition, other sensory stimulations, including tail pinches, were presented. Of the 52 units studied, 39 cells (75%) reacted to injections of hypertonic NaCl, but not the isotonic (control) solution (Normosol-R). Again, strength of neuronal reaction correlated with osmolarity of the NaCl solution injected, and force of injections was found not to influence results. Eleven cells reacted to hypertonic NaCl injections but not to tail pinch. This and other evidence indicated that certain dorsal midbrain cells were specifically osmosensitive, and not merely showing general 'arousal' reactions to the injections. These results indicate that, for the rat, the osmosensitive zone extends into the midbrain. The functional significance of these findings is discussed.     

Topographic organization and neurochemical identity of dorsal raphe neurons that project to the trigeminal somatosensory pathway in the rat.

The primary goals of this study were to: 1) examine the distribution of neurons within the dorsal raphe (DR) nucleus that project to cortical and subcortical sites along the trigeminal somatosensory pathway in rat; 2) determine the extent to which different regions within this ascending sensory system receive collateral projections from the same DR neuron; and 3) identify the putative transmitters contained within these DR projection neurons. Long-Evans hooded rats received pressure injections of various combinations of retrograde fluorescent tracers; into the whisker-related regions of the primary somatosensory cortex (barrel field cortex [BC]), ventral posterior medial thalamus (VPM), and principal nucleus of the trigeminal complex (PrV). The distribution of retrogradely labeled neurons within the DR was examined by fluorescence microscopy. The major finding was that cortically projecting neurons were located within the midline regions of the rostral portion of the DR, whereas cells projecting to subcortical trigeminal somatosensory structures were distributed bilaterally in the lateral wing regions of the DR as well as in the midline portions of the nucleus. Single neurons that send axon collaterals to multiple cortical and subcortical trigeminal somatosensory targets were observed in the dorsomedian and ventromedian regions of the DR. DR neurons that projected to cortical and subcortical sites contained serotonin but not tyrosine hydroxylase, the marker enzyme for catecholamine transmitters. Taken together, these findings provide further evidence of neurochemical specificity and functional anatomical organization within the DR efferent projection system.     

Hypothalamic and other connections with dorsal CA2 area of the mouse hippocampus

The CA2 area is an important, although relatively unexplored, component of the hippocampus. We used various tracers to provide a comprehensive analysis of CA2 connections in C57BL/6J mice. Using various adeno‐associated viruses that express fluorescent proteins, we found a vasopressinergic projection from the paraventricular nuclei of the hypothalamus (PVN) to the CA2 as well as a projection from pyramidal neurons of the CA2 to the supramammillary nuclei. These projections were confirmed by retrograde tracing. As expected, we observed CA2 afferent projections from neurons in ipsilateral entorhinal cortical layer II as well as from bilateral dorsal CA2 and CA3 using retrograde tracers. Additionally, we saw CA2 neuronal input from bilateral medial septal nuclei, vertical and horizontal limbs of the nucleus of diagonal band of Broca, supramammillary nuclei (SUM), and median raphe nucleus. Dorsal CA2 injections of adeno‐associated virus expressing green fluorescent protein revealed axonal projections primarily to dorsal CA1, CA2, and CA3 bilaterally. No projection was detected to the entorhinal cortex from the dorsal CA2. These results are consistent with recent observations that the dorsal CA2 forms disynaptic connections with the entorhinal cortex to influence dynamic memory processing. Mouse dorsal CA2 neurons send bilateral projections to the medial and lateral septal nuclei, vertical and horizontal limbs of the diagonal band of Broca, and SUM. Novel connections from the PVN and to the SUM suggest important regulatory roles for CA2 in mediating social and emotional input for memory processing. J. Comp. Neurol. 521:1844–1866, 2013. © 2012 Wiley Periodicals, Inc.     

Neurokinin 1 receptor expression by neurons in laminae I, III and IV of the rat spinal dorsal horn that project to the brainstem

Large neurons in laminae III and IV of the spinal cord which express the neurokinin 1 receptor and have dendrites that enter the superficial laminae are a major target for substance P (SP)-containing (nociceptive) primary afferents. Although some of these neurons project to the thalamus, we know little about other possible projection targets. The main aim of this study was to determine whether all cells of this type are projection neurons and to provide information about brainstem sites to which they project. Injections of cholera toxin B subunit were made into four brainstem areas that receive input from the spinal cord, and the proportion of cells of this type in the L4 spinal segment that were retrogradely labelled was determined in each case. The results suggest that most of these cells (>90%) project to the contralateral lateral reticular nucleus (or to a nearby region), while many (>60%) send axons to the lateral parabrachial area and some to the dorsal part of the caudal medulla. However, few of these cells project to the periaqueductal grey matter. As lamina I neurons with the neurokinin 1 receptor appear to be important in the generation of hyperalgesia, we also examined projection neurons in this lamina and found that for each injection site the great majority possessed the receptor. These results demonstrate that dorsal horn neurons which express the neurokinin 1 receptor contribute to several ascending pathways that are thought to be important in pain mechanisms.     

'Non-specific' cholinesterase-containing neurons of the dorsal thalamus project to medial limbic cortex

Thalamocortical neurons that contain 'non-specific' cholinesterase (ChE) were studied with cholinesterase histochemistry and experimental axonal tracing techniques in adult rats. In addition to the presence of ChE that is ubiquitous in capillary endothelium, neurons that contain ChE are found in 3 distinct regions of the dorsal thalamus, the thalamic reuniens nucleus (Re), the anterior dorsal nucleus (AD) and a region that includes the lateral part of the central lateral nucleus (CL) and the ventral portion of the lateral dorsal nucleus (LD). ChE activity appears light in cerebral cortex in general but histochemical staining is slightly greater in neuropil of the cingulate gyrus. Anterograde transport techniques with autoradiography demonstrated that neurons in the LD-CL region project to anterior cingulate cortex and the dorsal retrosplenial area. Anterograde degeneration techniques demonstrated that AD projects primarily to ventral retrosplenial cortex. Injections of horseradish peroxidase (HRP) in the anterior cingulate cortex resulted in double labeled cells (cells containing both ChE and HRP reaction products) primarily in LD and CL. HRP injections into ventral retrosplenial cortex resulted in double labeled cells in AD and Re. HRP injections in the subiculum resulted in double labeled cells in Re. Lesions placed in the region of thalamocortical projections resulted in a loss of ChE in the ipsilateral cingulate gyrus, as measured both histochemically and enzymatically. The finding that neurons containing ChE project to medial limbic cortex suggests that the ChE may be involved in the function of the thalamocortical component of the limbic system.     

The dorsal cochlear nucleus of the mouse: a light microscopic analysis of neurons that project to the inferior colliculus

In the mouse dorsal cochlear nucleus (DCN), all members of a distinct class of large multipolar neurons were shown to project to the contralateral inferior colliculus by using retrograde horseradish peroxidase techniques. Typically, these multipolar neurons have the largest cell bodies in the nucleus and are distributed in layers II, III, and IV. Each contains a round, pale nucleus with a prominent nucleolus and conspicuous Nissl bodies. In Golgi preparations, however, two types of large cells could be distinguished on the basis of dendritic characteristics. Pyramidal cells form relatively flattened, slablike dendritic fields whose alignment contributes to the laminar organization of the DCN. They represent 75-80% of the large cell population and are found in layer II and the superficial region of layer III. Giant cells represent the other type of large multipolar neuron and are distributed in the deeper regions of layer III and in layer IV. Their ellipsoidal dendritic fields are formed by long and relatively unbranched dendrites that project across the laminae. The differences in dendritic morphology imply that each cell class segregates its afferent input in distinct ways and subserves different auditory functions.     

Pituitary adenylate cyclase-activating polypeptide neurons of the ventromedial hypothalamus project to the midbrain central gray

The pituitary adenylate cyclase-activating polypeptide (PACAP) and brain-derived neurotrophic factor (BDNF) neurons have recently been established as markers of the ventromedial hypothalamic nucleus (VMH). However, their neural projections from the VMH remained unknown. We examined whether PACAP and BDNF neurons in the VMH connected to the mesencephalic central gray (MCG), using the combination of in-situ hybridization and immunohistochemical tracing of Fluorogold (FG) injected into the MCG. Approximately 49% of PACAP neurons and 19% of BDNF neurons in the VMH contained the retrograde-transported FG, and 52% of FG-positive cells were PACAP neurons. These results indicate that a large number of PACAP neurons and a small number of BDNF neurons of the VMH project to the MCG.     

Hypothalamic self-stimulation in rats with one hemisphere isolated anterior to the midbrain and the other hemisphere devoid of the telencephalon

The telencephalon was removed unilaterally in rats. In addition, the forebrain of the other hemisphere was isolated from its brain stem by a precollicular transverse cut. This preparation was then tested for self-stimulation via electrodes implanted in the region of the lateral hypothalamus and medial forebrain bundle (LH-MFB) of either hemisphere. Self-stimulation was found to be intact in both hemispheres. Possible pathways connecting the LH-MFB region of one hemisphere to the contralateral diencephalon were also investigated in the bilaterally detelencephalized rat. The technique of retrograde transport of horseradish peroxidase was used for this purpose. Some fibers from the LH-MFB of one hemisphere were found to decussate to the other hemisphere by way of the thalamic commissure. The supraoptic decussation as well as diffusely organized interdiencephalic connections provide additional routes for interhemispheric LH-MFB projections.     

GABAergic and other noncholinergic basal forebrain neurons,together with cholinergic neurons,project to the mesocortex and isocortex in the rat

The extrathalamic relay from the brainstem reticular formation to the cerebral cortex in the basal forebrain has been thought to be constituted predominantly, if not exclusively, by cholinergic neurons. In contrast, the septohippocampal projection has been shown to contain an important contingent of γ-aminobutyric acid (GABA)ergic neurons. In the present study, we investigated whether GABAergic neurons also contribute to the projection from the basal forebrain to neocortical regions, including the mesocortex (limbic) and the isocortex in the rat. For this purpose, retrograde transport of cholera toxin (CT) was examined from the medial prefrontal cortex for the mesocortex and from the parietal cortex for the isocortex and was combined with dual-immunohistochemical staining for either choline acetyltransferase (ChAT) or glutamic acid decarboxylase (GAD) in adjacent series of sections. Retrogradely labelled GAD⁺ neurons were codistributed with retrogradely labelled ChAT⁺ neurons through the basal forebrain from both the prefrontal and the parietal cortex, suggesting parallel, widespread cortical projections. The GAD⁺ cortically projecting cells were similar in size to the ChAT⁺ cells, thereby indicating that they comprise a contingent of the magnocellular basal cell complex. The proportions of retrogradely labelled neurons that were GAD⁺ (approximately one-third) were equal to or greater than those that were ChAT⁺ from both the prefrontal cortex and the parietal cortex. In addition, the total of GAD⁺ and ChAT⁺ neurons did not account for the total number of cortically projecting cells, indicating that another equivalent proportion of chemically unidentified noncholinergic neurons also contributes to the basalocortical projection. Accordingly, as in the allocortex, GABAergic, cholinergic, and other unidentified noncholinergic neurons may have the capacity to modulate activity in the mesocortex (limbic) and the isocortex through parallel, widespread projections. J. Comp. Neurol. 383:163-177, 1997. © 1997 Wiley-Liss, Inc.     

Hypothalamic afferents to the dorsal dentate gyrus contain acetylcholinesterase

Retrograde transport of Evans blue dye from the rat dentate gyrus was used to identify afferent hypothalamic cells. Photography of the fluorescent hypothalamic cells was followed by incubation in an acetylthiocholine medium. Slides were re-photographed for acetylcholinesterase-induced reaction product. Nearly all posterior hypothalamic cell afferents to the dentate gyrus could be positively identified as containing acetylcholinesterase.     

Galanin/alpha2-adrenoceptor interactions in telencephalic and diencephalic regions of the rat

The aim of this study was to evaluate whether galanin could affect central alpha2-adrenoceptors in telencephalic and diencephalic regions in the rat using quantitative receptor autoradiography with the alpha2 agonist radioligand [3H]p-aminoclonidine. Galanin 1 nM significantly and substantially increased the Kd value of the [3H]p-aminoclonidine binding sites in the medial hypothalamus and amygdala by 86% (p < 0.01) and 73% (p < 0.05) respectively. The Bmax value was only significantly increased with 3 nM galanin in the amygdala and the medial hypothalamus (both p < 0.05). The antagonist M35 counteracted the increase of the Kd values of the alpha2-adrenoceptor agonist binding sites produced by galanin 1 nM in the amygdala and the medial hypothalamus (both p < 0.001). These findings suggest the existence of an antagonistic galanin/alpha2 adrenoceptor interaction in the medial hypothalamus and amygdala that may be of relevance for alpha2-adrenoceptor-regulated neuroendocrine functions and food intake.     

Responses of diencephalic neurons to sensory stimulation in the goldfish, Carassius auratus

We studied the responses to sensory stimulation in two diencephalic areas, the central posterior nucleus of the dorsal thalamus (CP) and the anterior tuberal nucleus of the hypothalamus (TA). In both the CP and the TA, units sensitive to acoustic (500-Hz sound), hydrodynamic (25-Hz dipole stimulus), and visual (640-nm light flash) stimuli were found. In the CP, most units were unimodal and responded exclusively to visual stimulation. In contrast, in the TA, most units responded to more than one modality. The data suggest that the CP is primarily involved in the unimodal processing of sensory information, whereas the TA may be involved in multisensory integration.     

Calbindin-immunoreactive sensory neurons of dorsal root ganglion project to skeletal muscle in the chick

In the chicken dorsal root ganglia, two neuronal subpopulations referred to as A1 and B1 share in common an immunoreactivity to antisera raised to calbindin D-28k but are distinguished by their cytological and ultrastructural characteristics. To determine the peripheral targets innervated by calbindin-immunoreactive neurons in lumbosacral dorsal root ganglia, cryostat sections of various hindlimb tissues were treated with anticalbindin antisera. Calbindin-immunostained axons were clearly detected in skeletal muscle. Large myelinated nerve fibres and afferent axon terminals in neuromuscular spindles were calbindin-immunoreactive; thin unmyelinated nerve fibres were also immunostained in nerve bundles of the perimysium. Since motoneurons and neurons of the autonomic nervous system were devoid of calbindin immunostaining, it was suggested that the immunoreactive axons found in skeletal muscle originate from sensory neurons expressing a calbindin immunoreaction in the dorsal root ganglia. This hypothesis was corroborated after introduction of wheat germ agglutinin coupled with horseradish peroxidase or colloidal gold particles into the sartorius muscle. The retrogradely transported tracer was collected only in ganglion cell bodies which displayed the ultrastructural characteristics of A1 and B1 sensory neurons. On the basis of calbindin immunoreaction and of tracer retrograde transport, it is concluded that ganglion cells of subclasses A1 and B1 contribute to the sensory innervation of skeletal muscle in the chicken.     

Systemic oxytocin treatment modulates alpha 2-adrenoceptors in telencephalic and diencephalic regions of the rat

Systemic subchronic oxytocin treatment significantly and substantially increased the B(max) values of the alpha 2 agonist [(3)H]UK14.304 binding sites in the hypothalamus, the amygdala and the paraventricular thalamic nucleus of the rat as shown by quantitative receptor autoradiography. These results suggest that long-term modulation of autonomic and neuroendocrine functions and emotional behaviours elicited by brain oxytocin may involve enhancement of central alpha 2-adrenoceptor function.     

Topographic organization of neurons in the acoustic thalamus that project to the amygdala

Projections from the posterior thalamus to the amygdala have been implicated in the processing of the emotional significance of acoustic stimuli. The aim of the present studies was to determine which areas of the amygdala receive afferents from posterior thalamic structures that, in turn, receive afferents (presumably acoustic afferents) from the inferior colliculus. Projections from the posterior thalamus to the amygdala and striatum were examined in rats using anterograde and retrograde axonal transport techniques. Following injections of WGA-HRP into the posterior thalamic areas [including the medial division of the medial geniculate body, the posterior intralaminar nucleus (PIN) and the medial posterior complex (POM)], anterograde transport was seen in the lateral (AL), central (ACE), medial (AM), and basomedial (ABM) nuclei of the amygdala and in the amygdalostriatal transition area (AST) and posterior caudate putamen (CPU). Injection of WGA-HRP into each anterogradely labeled area produced retrograde transport to the posterior thalamus, but the pattern of transport varied with the site of the injection. Injections in AL and AST produced retrograde transport to neurons in the medial division of the medial geniculate body (MGM), PIN, suprageniculate nucleus (SG) and, to a lesser extent, the lateral posterior nucleus (LP). Injections of the ACE, AM, and ABM, in contrast, only labeled cells in POM. While the MGM, PIN, and SG each receive afferents from the inferior colliculus, POM does not. AL and AST, therefore, receive inputs from thalamic areas that, in turn, receive inputs from the inferior colliculus.     

Hypothalamic glioma with diencephalic syndrome and following precocious puberty--a case report

A 1-year 11-month-old girl was admitted for losing her weight and gait disturbance. At 4 months of age, she began to become emaciated inspite of normal food intake. Physical and neurological examinations were normal except for a marked lack of subcutaneous fat, irritability and nystagmus. CT scans demonstrated a large tumor occupied in the third ventricle and marked dilatation of the lateral ventricles. Endocrinological studies revealed high levels of plasma growth hormone (GH) in contrast with normal levels of somatomedin-C. The basal value of GH returned to normal with a subnormal response to insulin subsequently after VP-shunt. Then, a pilocytic astrocytoma was partially resected with transcallosal approach. Postoperative course was uneventful and her growth rate returned to normal range. CT scans after radiation therapy of 49 Gy showed marked decrease in size of the tumor. At 3 years and 6 months of age, enlargement of her breast was pointed out although MRI indicated no enlargement of the tumor. Basal value of LH, FSH, E 1 and E 2 elevated and LHRH test showed over-response of LH and FSH. Other hypothalamic-pituitary functions were partially preserved. Case of precocious puberty following diencephalic syndrome associated to the hypothalamic and/or optochiasmatic glioma is quite rare in the previous literature. Mechanisms of diencephalic syndrome and following puberty are unclear. However, endocrinological and radiological findings observed in the present case suggest that hormonal disfunction might be due to the failure of inhibition on GH and LHRH secretion mechanism in the anterior hypothalamus.