Extranuclear axon collaterals of paraventricular neurons in the rat hypothalamus: Intracellular staining, immunocytochemistry and electrophysiology

Recent studies have suggested that some paraventricular nucleus (PVN) neurons projected to more than one target and, thereby, perhaps coordinate some aspects of seemingly diverse functions. We have systematically investigated the existence, location, hormonal contents and functional integrity of some axon collaterals arising from PVN neurons. This was done using intracellular injections of the fluorescent dye, Lucifer Yellow, extracellular ejections of horseradish peroxidase (HRP), immunocytochemistry with antisera directed against vasopressin (VP) and oxytocin (OX) and electrophysiological analysis of synaptic activation of perifornical neurons in response to electrical stimulation of the PVN in hypothalamic slices. Each of the three morphological techniques revealed clear axon collaterals, arising in the lateral hypothalamus and generally ventrolateral to the PVN. Most branching axons appeared to have a small number of branch points, and many collaterals appeared to terminate near their parent axon. Electrical stimulation of the PVN was found to activate synaptically perifornical neurons located in the areas where the other methods revealed collaterals. Stimulation outside of the nucleus was ineffective unless current intensities were increased 10-30-fold over those applied to the PVN. We conclude that many PVN neurons, at least some of these containing OX and other VP, give rise to axons that branch in the perifornical and more ventral lateral hypothalamus, and that some of their collaterals probably terminate on neurons close to the PVN.     

Basal forebrain neurons have axon collaterals that project to widely divergent cortical areas in the cat

Basal forebrain neurons with axon collaterals that project to widely divergent cortical areas were identified using retrograde transport of two labels. A proportion of neurons in the basal forebrain have axon collaterals that project to both anterior (precruciate gyrus) and posterior (marginal and suprasylvian gyri) cortical areas or to medial (precruciate gyrus) and lateral (ectosylvian and anterior suprasylvian gyri) cortical areas. These branched fibers originate from cells located predominantly in the basal nucleus of Meynert. The existence of such neurons suggests that individual basal forebrain cells are capable of influencing widespread neocortical zones in the cat.     

Electrophysiological evidence favoring intracaudate axon collaterals of GABAergic caudate output neurons in the cat

Stimulation of the entopeduncular nucleus in the cat was shown to evoke inhibitory responses with a short onset-latency in the caudate nucleus isolated from its afferents. These inhibitory responses are shown to be GABAergic, and some of them are suggested to be monosynaptic in nature.     

Raphespinal and reticulospinal axon collaterals to the hypoglossal nucleus in the rat.

Neurons in the medial tegmental field project directly to spinal somatic motoneurons and to cranial motoneuron pools such as the hypoglossal nucleus. The axons of these neurons may be highly collateralized, projecting to multiple levels of the spinal cord and to many diverse regions at different levels of the neuraxis. We employed a double fluorescent retrograde tracer technique to examine whether medial tegmental neurons that project to the spinal cord also project to the hypoglossal nucleus. Injections of Diamidino Yellow into the hypoglossal nucleus and Fast Blue into the spinal cord produced large numbers of double labeled neurons in the medial tegmental field, particularly in the caudal raphe nuclei and adjacent ventromedial reticular formation. In these structures the number of neurons projecting to both the hypoglossal nucleus and the spinal cord was equivalent to the number of neurons projecting to multiple levels of the spinal cord observed in control animals. Fewer neurons projecting to both the hypoglossal nucleus and the spinal cord were observed in several other nuclei and subregions of the medial tegmental field, while almost no such neurons were observed in the lateral tegmental field or other pontomedullary structures. These results demonstrate that neurons of the caudal raphe nuclei and adjacent ventromedial reticular formation project to both the spinal cord and the hypoglossal nucleus, and support the concept that the diffuse projections to motoneuron pools from the medial tegmental field globally modulate both spinal and cranial somatic motoneuron excitability.     

Olivocochlear neurons sending axon collaterals into the ventral cochlear nucleus of the rat

The olivocochlear projection constitutes the last stage of the descending auditory system in the mammalian brain. Its neurons reside in the superior olivary complex (SOC) and project to the inner and outer hair cell receptors in the cochlea. Olivocochlear neurons were also reported to send axon collaterals into the cochlear nucleus, but controversies about their number and about species differences persist. By injecting the fluorescent retrograde axonal tracers diamidino yellow and fast blue into the cochlea and the ventral cochlear nucleus (VCN), we studied the distribution and number of olivocochlear neurons with and without axon collaterals into the VCN of the rat. We found that olivocochlear neurons residing in the lateral superior olive (LSO), the intrinsic lateral olivocochlear cells (intrinsic LOCs), do not send axon collaterals into the VCN. By contrast, a majority, and possibly all, olivocochlear neurons residing in the ventral nucleus of the trapezoid body (VNTB), the medial olivocochlear cells (MOCs), do have such axon collaterals. These cells may thus affect processing in the ascending auditory pathway at the level of the receptors and concurrently at the level of the secondary sensory neurons in the cochlear nucleus. Belonging to the lateral olivocochlear system, shell neurons reside around the LSO and form a third group of olivocochlear cells (shell LOCs). Like intrinsic LOCs, they innervate the inner hair cells, but like MOCs they do, by means of axon collaterals, project into the VCN. These findings have implications for understanding both auditory signal processing and the plasticity responses that occur following loss of cochlear function.     

Organization of local axon collaterals of efferent projection neurons in rat visual cortex

We have studied the laminar origins of local long-range connections within rat primary visual cortex (area 17), by using retrograde tracing of nerve cell bodies with fluorescent markers. Injections throughout the thickness of cortex produce distinct laminar labeling patterns which indicate that a substantial number of cells in layers 2/3, 5, and 6 have wide local axon collateral arbors, while the local arbors of layer 4 cells are much narrower. Double labeling experiments which combined area 17 injections with injections into different projection targets of area 17 (opposite area 17, area 18a, and area 18b) show that many cortico-cortically projecting cells make widespread projections within area 17. In contrast, the overwhelming majority of subcortically projecting cells have narrow collateral arbors within area 17. Anterograde tracing of local projections within areas 17 with the lectin Phaseolus vulgaris leucoagglutinin shows an extensive system of horizontally running fibers which terminate in distinct 0.15-0.25 mm wide clusters up to 1.8 mm from the injection site. On horizontal sections the termination pattern resembles a closely spaced lattice. The results indicate that cortico-cortically projecting cells provide for long-range interactions between distant points of the visuotopic map, while subcortically projecting cells mediate information within a cortical column. Interestingly, subcortically projecting cells differ functionally from cortico-cortically projecting cells in that they are not orientation selective (Klein et al., Neurosci. 17:57-78, '86; Mangini and Pearlman, J. Comp. Neurol. 193:203-222, '80; Simmons and Pearlman, J. Neurophysiol. 50:838-848, '83). We therefore suggest that cortico-cortically projecting cells with wide collateral arbors are orientation selective and that clustered long-range projections within area 17 connect columns with similar functional specificity.     

Anatomical distribution of LHRH-immunoreactive neurons in the human infant hypothalamus and extrahypothalamic regions

The morphological features and distribution of luteinizing hormone-releasing hormone (LHRH)-immunoreactive cell bodies and fibers of the hypothalamic and the neighboring mesencephalic regions were studied in the normal newborn infant by immunohistochemistry. Within the hypothalamus, numerous LHRH-immunoreactive like (IL) cell bodies were found mainly in the ventral portion of the infundibular nucleus close to the median eminence and at a lower extent in the medial preoptic area. In addition, sparse immunoreactive cell bodies were displayed in the paraventricular and medial mammillary nuclei. The mesencephalon also exhibited rare immunoreactive cell bodies in the periaqueductal gray. LHRH-IL fibers, predominantly varicose, formed a continuum from the septo-preoptico level to the mesencephalon. In the hypothalamus, the median eminence exhibited the highest LHRH innervation. LHRH-IL fibers are also observed in the lamina terminalis, the medial preoptic area, the suprachiasmatic, the supraoptic, the peri- and the paraventricular nuclei. In the last two nuclei, some fibers projected to the dorsomedial and ventromedial nuclei whereas others were in close relation with the ependyma. The mesencephalon displayed low LHRH-IL fibers, present essentially in the raphe and interpeduncular nuclei and around the ependyma. When compared with data obtained in other mammals, the present findings agree well with the general distribution and morphological features of LHRH-IL neuronal structures reported elsewhere.     

Subfornical organ neurons with efferent projections to the hypothalamic paraventricular nucleus: an electrophysiological study in the rat

Seventeen neurons in the subfornical organ (SFO) were antidromically activated by electrical stimulation of the paraventricular nucleus (PVN) in the rat. The activity of all identified SFO neurons was excited by microiontophoretically (MIPh) applied angiotensin II (AII) and the effect of AII was blocked by MIPh-applied saralasin (Sar), an AII antagonist, but not by atropine (Atr), a muscarinic antagonist. In these identified SFO neurons, 9 were also excited and 8 were not affected by MIPh-applied acetylcholine (ACh) and the effect of ACh was attenuated by not only MIPh-applied Atr but also by Sar. These results suggest that there are specific AII- and both AII- and ACh-sensitive types of SFO neurons with efferent projections to the PVN.     

Topography of serotonin neurons in the dorsal raphe nucleus that send axon collaterals to the rat prefrontal cortex and nucleus accumbens

Diverse physiological actions have been reported for 5-hydroxytryptamine (5-HT, serotonin) in the medial prefrontal cortex (MPFC) and the nucleus accumbens (Acb) suggesting that the 5-HT innervation of these forebrain areas may be derived from different populations of neurons. We examined this possibility by mapping the distribution of 5-HT-immunoreactive (ir) and non-5HT-ir neurons containing retrograde labeling following injections of different tracers into both these target regions. The analysis was focused in the dorsal raphe nucleus (DRN) of the midbrain, since 5-HT pathways to the MPFC and Acb primarily originate from this area. Volume microinjections of the fluorescent retrograde tracer, Fluoro-Gold (FG), were placed into the MPFC and microinjections of cholera toxin B subunit coupled to 15 nm gold particles (CT-Au) were placed into the Acb of the same animal. Sections through the DRN containing retrogradely labeled neurons were further processed for immunofluorescent localization of 5-HT using a rhodamine marker. Neurons retrogradely labeled from the Acb were greater in number overall than those projecting to the MPFC. In addition, Acb-projecting neurons extended into the lateral wings of the DRN, whereas MPFC-projecting neurons were more restricted to the midline. Both groups of retrogradely labeled neurons, however, were more numerous in the caudal aspect of the dorsal raphe nucleus and were scattered amongst 5-HT immunoreactive perikarya. Of783 ± 69 CT-Au labeled cells, 15% also contained the FG label and 11% contained FG and 5-HT immunoreactivity. Of613 ± 48 FG labeled cells, 24% also contained the CT-Au label and 21% were also immunoreactive to 5-HT. The results suggest a more prominent input to the Acb from both 5-HT-ir and non-5-HT-ir neurons in the caudal aspect of the DRN and further indicate that while most 5-HT-ir and non-5-HT-ir neurons project differentially to both forebrain regions, a few cells also show collateralization to the MPFC and Acb. Such collateralization of single serotonergic neurons to divergent targets mey integrate cognitive and motor activities in response to pharmacological manipulations of ascending serotonergic pathways.     

Organization of tyrosine hydroxylase- and serotonin-immunoreactive brainstem neurons with axon collaterals to the periaqueductal gray and the spinal cord in the rat

Retrograde tracing and immunocytochemistry were used to examine the axon collateralization of brainstem serotonin (5-HT) and norepinephrine (NE) cells to the periaqueductal gray (PAG) and spinal cord. Tyrosine hydroxylase (TH)-immunofluorescent neurons which collateralize to the PAG and the cervical spinal cord were found in all brainstem catecholamine cell groups previously shown to contain neurons which project to the spinal cord, including the A5 and A7 cell groups, locus coeruleus, subcoeruleus and the C1 cell group. Many TH-immunofluorescent cells which project to the PAG but not to the spinal cord were also found. The region of the nucleus raphe magnus (NRM) also contained many neurons retrogradely labeled from the PAG. These overlapped with the distribution of spinally projecting 5-HT-immunofluorescent cells in the NRM, however, less than 1% of the PAG projecting cells in this region were 5-HT-immunofluorescent. In contrast, many 5-HT-immunofluorescent cells in the more rostral nucleus raphe pontis and nucleus raphe dorsalis were retrogradely labeled from the PAG but not from the spinal cord. Finally, a population of neurons in the NRM and adjacent reticular formation and in the region of several pontomedullary catecholamine cell groups collateralized to the PAG and spinal cord, but were neither 5-HT nor TH-immunofluorescent. Taken together, these findings raise the possibility that the noradrenergic contribution to the spinal antinociceptive effects produced by PAG electrical stimulation results, in part, from antidromic activation of brainstem noradrenergic neurons that have axon collaterals projecting to the PAG and spinal cord. In contrast, the 5-HT contribution to the spinal antinociceptive effects produced by PAG electrical stimulation is more likely to derive, as previously proposed, from orthodromic activation of raphe-spinal serotonergic axons.     

Spinal neurons which project to the periaqueductal gray and the medullary reticular formation via axon collaterals: a double-label fluorescence study in the rat

Fluorescent retrograde double-labeling methods were used in which Fast blue and Nuclear yellow or Diamidino yellow dihydrochloride were injected into the midbrain periaqueductal gray (PAG) and medullary reticular formation (MRF). Double-labeled neurons were most frequently observed in the lateral part of lamina V, in laminae VII, VIII and X and in the lateral cervical and lateral spinal nuclei. The data demonstrate that some spinal neurons project to both the PAG and the MRF via axon collaterals.     

Morphology of local axon collaterals of electrophysiologically characterised neurons in the rat medial septal/ diagonal band complex

Neurons in the medial septal/diagonal band complex (MS/DB) in vivo exhibit rhythmic burst-firing activity that is phase-locked with the hippocampal theta rhythm. The aim was to assess the morphology of local axon collaterals of electrophysiologically identified MS/DB neurons using intracellular recording and biocytin injection in vitro. Cells were classified according to previous criteria into slow-firing, fast-spiking, regular-spiking, and burst-firing neurons; previous work has suggested that the slow-firing neurons are cholinergic and that the other types are GABAergic. A novel finding was the existence of two types of burst-firing neuron. Type I burst-firing neurons had significantly longer duration after hyperpolarisation potentials when held at -60 mV, and at -75 mV, type I neurons exhibited a low-threshold spike with more rapid activation and inactivation kinetics than those of type II neurons. We have, also for the first time, described the main features of the local axon collaterals of the five neuron types. All filled neurons possessed a main axon that gave forth 1-12 local primary axon collaterals. All electrophysiological types, except for the type I burst-firing neuron, had a main axon that coursed toward the fornix. Myelination of the main axon was a prominent feature of all but the slow-firing neurons. Branching of the primary axon collaterals of the fast-spiking and type I burst-firing neurons was more extensive than that of the other cell types, with those of the slow-firing neurons exhibiting the least branching. All cell types possessed axon collaterals of the en passant type, and some in addition had twiglike or basketlike axon terminals. All cell types made synapses on distal dendrites; a proportion of the fast-spiking and burst-firing cells in addition had basketlike terminals that made synaptic contacts on proximal dendrites and on somata. Two morphological types of somata were postsynaptic to the basket cells: large (20-30-microm) oval cells with dark cytoplasm, and large oval cells with paler cytoplasm, often with an apical dendrite. The presence of lamellar bodies in the large dark neurons suggests that they may be cholinergic neurons, because previous work has localised these structures in some neurons that stain for choline acetyltransferase. Our work suggests therefore that there may be GABAergic neurons in the MS/DB that form basket synaptic contacts on at least two types of target cell, possibly cholinergic and GABAergic neurons, which means that the basket cells could play a key role in the generation of rhythmic activity in the MS/DB.     

Connections of hypothalamic paraventricular neurons with the dorsal medial thalamus and neurohypophysis: an electrophysiological study in the rat

Connections between the hypothalamic paraventricular nucleus (PVN) and thalmic paraventricular nucleus (PVT) were examined using electrophysiological methods. Efferent projections of adjacent PVN cells were defined on the basis of antidromic activation from either PVT (n= 12) or neurophyphoseal (n= 38) stimulation; antidromic activation from both sites (n= 3) suggested that some PVN cells project both to the PVT and to the neurohypophysis. PVT stimulation evoked only weak orthodromic responses from 21% of PVN neurohypophyseal neurons, whereas short latency, high probability orthodromic responses were observed from 43% of PVN non-neurosecretory neurons. These data indicate reciprocal PVN-PVT connections and suggest that PVT afferents preferentially innervate non-neurosecretory PVN cells.     

An electrophysiological study of inputs to neurons of the ventral tegmental area from the nucleus accumbens and medial preoptic-anterior hypothalamic areas

Extracellular recordings were obtained from neurons in the ventral tegmental area (VTA) of urethane-anesthetized rats. Neurons were devided into two types based on the latencies of antidromic activation following electrical stimulation of the nucleus accumbens (NAcc), and on the durations of action potentials. Type A neurons had longer latencies for antidromic activation (mean 15.9 msec) and longer durations of action potentials (2.6msec), while type B neurons had shorter latencies (mean 4.5 msec) and shorter duration of action potentials (< 2.6msec).Electrical stimulation of the medial preoptic-anterior hypothalamic areas (mPOA-AHA) and NAcc produced the following effects on the two types of VTA neurons: (i) the majority of both type A and B neurons were suppressed by mPOA-AHA stimula stimulation with onset latencies of less than 10 msec; (ii) 42% of type B neurons were also suppressed by NAcc stimulation, with onset latencies of less than 10 msec; (iii) type A neurons were suppressed (33%) or activated (43%) by NAcc stimulation, the onset latencies usually being longer than 10 msec; (iv) 71% of type A neurons tested had convergent inputs from the mPOA-AHA and NAcc, usually suppressed-suppressed or suppressed-activated, while 45% of type B neurons had convergent inputs from these two areas, usually suppressed-suppressed.     

Morphological and electrophysiological evidence for electrotonic coupling of rat dorsolateral septal nucleus neurons in vitro

Intracellular injections of Lucifer Yellow were utilized to evaluate the incidence of dye-coupling among dorsolateral septal nucleus (DLSN) neurons recorded from slice preparations of adult rat septal nuclei. Twenty percent of single injections of Lucifer Yellow resulted in pairs of labeled neurons. These dye-coupled cells were morphologically heterogeneous and did not exhibit any morphological characteristics that could be used to distinguish them from non dye-coupled neurons. The spatial separation of cell bodies and close apposition of dendrites within each pair indicated that the dye transfer site(s) were situated at dendrodendritic and/or dendrosomatic rather than somatosomatic junctions. The main axon of some dye-coupled neurons gave rise to intrinsic axon collaterals prior to exiting the nucleus indicating that these coupled neurons function as projection neurons as well as local circuit interneurons. Electrophysiological recordings of the passive membrane properties and spontaneous activity of individual dye-coupled neurons revealed no significant difference from non dye-coupled cells in the DLSN. Some neurons exhibited spontaneously occurring fast potentials which presumably represent electrotonic potentials. These fast potentials were often tightly coupled with action potentials but could be distinguished from synaptic potentials by their shape and their lack of voltage-dependent changes in amplitude. These morphological and supportive electrophysiological data provide the first indirect evidence for electrotonic coupling of dorsolateral septal neurons. The functional significance of this coupling may lie in the potential for synchronization of the output of the DLSN which could play an important role in the septal maintenance and modulation of hippocampal Theta rhythm. © 1993 Wiley-Liss, Inc.     

Anatomical and electrophysiological development of the hypothalamic orexin neurons from embryos to neonates

The amount, quality, and diurnal pattern of sleep change greatly during development. Developmental changes of sleep/wake architecture are in a close relationship to brain development. The fragmentation of wake episodes is one of the salient features in the neonatal period, which is also observed in mature animals and human individuals lacking neuropeptide orexin/hypocretin signaling. This raises the possibility that developmental changes of lateral hypothalamic orexin neurons are relevant to the development of sleep/wake architecture. However, little information is available on morphological and physiological features of developing orexin neurons. To address the cellular basis for maturation of the sleep/wake regulatory system, we investigated the functional development of orexin neurons in the lateral hypothalamus. The anatomical development as well as the changes in the electrophysiological characteristics of orexin neurons was examined from embryonic to postnatal stages in orexin‐EGFP mice. Prepro‐orexin promoter activity was detectable at embryonic day (E) 12.0, followed by expression of orexin A after E14.0. The number of orexin neurons and their membrane capacitance reached similar levels to adults by postnatal day (P) 7, while their membrane potentials, firing rates, and action potential waveforms were developed by P21. The hyperpolarizing effect of serotonin, which is a major inhibitory signal for adult orexin neurons, was detected after E18.0 and matured at P1. These results suggest that the expression of orexin peptides precedes the maturation of electrophysiological activity of orexin neurons. The function of orexin neurons gradually matures by 3 weeks after birth, coinciding with maturation of sleep/wake architecture.     

The rat striatum: a target nucleus for ascending axon collaterals of the entopedunculo-habenular pathway

Direct projections from the entopeduncular nucleus (Ep) to the striatum were examined in the rat using retrograde tracing techniques. After injecting horseradish peroxidase (HRP) into the caudoputamen (CPU), labeled fibers could be traced medially to go through and/or terminate in both the globus pallidus (GP) and Ep ipsilateral to the injection. Interestingly, HRP-positive neuronal perikarya were observed in the Ep as well as in the GP after the CPU injections. These labeled Ep neurons were medium in size and restricted to the rostral 1/3 of the nucleus, in a distribution which overlapped the terminal fields from the CPU. A fluorescent tracer injection into the CPU resulted in retrograde labeling of Ep cells in the same fashion as after HRP injections. A second experiment was designed to determine whether the Ep cells projecting to the CPU have axon collaterals to any of the other known terminal fields of the Ep. Following True blue (TB) injections into the CPU and Diamidino yellow (DY) injections into the lateral habenular nucleus (lHb), all the TB-positive neurons in the rostral 1/3 of the Ep were unequivocally double-labeled with DY. On the other hand, the TB-labeled Ep neurons were never double labeled with DY injected into the centre median-parafascicular complex (CM-Pf) or ventroanterior-ventrolateral complex (VA-VL) of the thalamus or nucleus tegmenti pedunculopontinus pars compacta (TPC). The present study shows that single Ep neurons in the rostral 1/3 of the nucleus innervate both the striatum and lHb, and raises the possibility that these neurons may have a critical role in integrating motor and limbic functions in the basal ganglia.     

Further evidence for endogenous hypothalamic serotonergic neurons involved in the cimetidine-induced prolactin release in the rat

The aim of the present work was to further explore the possible relationship between the prolactin-releasing effect of cimetidine and hypothalamic serotonergic neurons controlling pituitary hormone secretion. In a first approach, the prolactin-releasing effect of the drug was determined in adult male rats with total deafferentation of the hypothalamus. Cimetidine injection (60 mg/kg) produced a significant rise in prolactin, but not in luteinizing hormone (LH), both in deafferented rat and in sham-operated controls; by 15 min there was a 5-6 fold increase in prolactin titers. Methysergide, a serotonin receptor blocker, used in a dose (2.5 mg/kg), route (i.p.) and time (50 min earlier) which did not modify the hormone basal level in rats with total deafferentation of the hypothalamus, was able to prevent completely the prolactin release evoked by cimetidine. The same preventive effect on prolactin release was observed with the serotonin receptor blocker ketanserin (5 mg/kg, i.p., 30 min earlier). It is concluded that the prolactin-releasing effect of cimetidine is located at a hypothalamic level related to serotonergic neurons.