Fasciculus Retroflexus Research Articles

Ontogeny of ( d-Ala 2)-deltorphin I-like immunoreactive neurones in foetal rat brain

Foetal rat brain from embryonic day (ED) 12–22 was immunohistochemically studied to describe the time of first appearance and further distribution patterns of ( d-Ala 2)-deltorphin-I-immunoreactive (DADTI-IR) nerve elements. The primary antiserum used in this study was a polyclonal antibody against DADTI previously used in adult and postnatal rat brain mapping. DADTI-IR nerve elements first appeared in the neuroepithelium of ventral mesencephalon on ED 13. From there, positive cell bodies migrated towards the mantle layer until they invaded the whole ventral mesencephalic tegmentum. They then reached their definitive position, corresponding to a subpopulation of the A8, A9 and A10 dopaminergic neurones that had been constantly observed also in the adult age. From ED 15–17, DADTI-positive nerve fibres appeared in the medial forebrain bundle, the neostriatum anlage, the accumbens nucleus, the olfactory tubercle, the fasciculus retroflexus, and the prefrontal cortex. All these locations have also been found in adult rats. From ED 14 onwards, transient DADTI-IR somata and nerve fibres were observed in retinal neuroepithelium, optic pathways as far as the superior colliculus, CA3 hippocampal field, reticular formation in the medulla oblongata. All these locations gradually disappeared either before birth (medulla oblongata) or within the first 3 weeks after birth. These results suggest that the DADT-like molecule recognised by our antibody has during the embryonic development a regulatory function in neuronal growth and differentiation.

Expression of the Netrin-1 receptor, deleted in colorectal cancer (DCC), is largely confined to projecting neurons in the developing forebrain

Axon guidance mechanisms are crucial to the development of an integrated nervous system. One family of molecules that may be important in establishing axonal connectivity in mammals is the Netrins, and their putative receptors DCC (deleted in colorectal cancer), Neogenin, and Unc-5. Knockout and mutational analyses of some of these genes have shown that they are critically involved in the development of several specific pathways in the developing brain. However, previous expression analyses of these genes have largely been confined to the developing spinal cord. In the present study, we analyzed the expression of DCC in the developing mouse forebrain. We found that DCC protein is expressed in specific axonal populations projecting from the developing olfactory bulb, neocortex, hippocampus, and epithalamus/habenular complex. In the developing olfactory bulb and neocortex, DCC expression is particularly evident during the targeting phase of axon outgrowth and is then rapidly downregulated. As predicted from the knockout and mutational analyses of this gene, DCC is expressed in axonal commissures, in particular the corpus callosum, hippocampal commissure, and the anterior commissure. In addition, we found that DCC is expressed in the habenular commissure, the fasciculus retroflexus, and the stria medularis. Therefore, this analysis implicates a function for DCC in additional axonal guidance systems not predicted from the knockout and mutational analyses.

Distribution of choline acetyltransferase (ChAT) immunoreactivity in the central nervous system of a chondrostean, the siberian sturgeon (Acipenser baeri).

All studies to date of cholinergic systems of bony fishes have been done in teleosts. To gain further insight into the evolution of the cholinergic systems of bony fishes, we have studied the brain of a chondrostean fish, the Siberian sturgeon (Acipenser baeri, Brandt), by using an antibody against choline acetyltransferase (ChAT). This study showed the presence of ChAT-immunoreactive (ChAT-ir) neurons in the preoptic region (parvocellular and magnocellular preoptic nuclei and suprachiasmatic nucleus), the periventricular and tuberal hypothalamus, the saccus vasculosus, the dorsal thalamus, and the habenula. The mesencephalic tegmentum contained ChAT-ir cells in the torus semicircularis and torus lateralis. The isthmus contained several cholinergic populations: the nucleus isthmi, the lateral nucleus of the valvula, the secondary visceral nucleus, and the dorsal tegmental nucleus. The motor neurons of the cranial nerves and the spinal motor column were strongly immunoreactive. The medial (sensory) trigeminal nucleus also contained a ChAT-ir neuronal population. The distribution of ChAT-ir neurons in the sturgeon brain showed some notable differences with that observed in teleosts, such as the absence of cholinergic cells in the telencephalon and the optic tectum. Several brain regions were richly innervated by ChAT-ir fibers, particularly the telencephalon, optic tectum, thalamus, posterior tubercle, and interpeduncular nucleus. The hypothalamo-hypophyseal tract, the tract of the saccus vasculosus, the fasciculus retroflexus, and an isthmo-mesencephalo-thalamic tract were the most conspicuous cholinergic bundles. Comparative analysis of these results suggests that teleosts have conserved most traits of the cholinergic system of the sturgeon, having acquired new cholinergic populations during evolution.

Prenatal cocaine produces signs of neurodegeneration in the lateral habenula

The lateral habenula is a nucleus in the dorsal thalamus that innervates midbrain dopaminergic and serotonergic nuclei via projections through its major efferent pathway, the fasciculus retroflexus (FR). It was previously demonstrated that cocaine administered continuously to adult rats over several days produces neurodegeneration in the lateral habenula and FR. Because exposure to cocaine during pregnancy reportedly can cause neurobehavioral deficits, we examined whether rat fetuses exposed to continuous cocaine during the last week of gestation would similarly demonstrate selective neurodegeneration in the lateral habenula. On day 17 of gestation, dams were implanted with two silicone pellets, each containing either vehicle or one of 2 doses of cocaine (80 mg or 55 mg per pellet). Degenerating neurons containing silver deposits were counted in lateral habenula and in the striatum. Cocaine-exposed pups had significantly more silver-stained cells in the lateral habenula than vehicle-treated pups, but similar numbers of silver-stained cells were present in the striatum of all three groups. When similarly treated vehicle- and cocaine-exposed animals were tested behaviorally at 60 days of age, they did not differ on measures of open field activity, open arm avoidance on the elevated plus-maze or conditioned place preference for cocaine, although a linear trend analysis indicated some hyperactivity of the cocaine-pretreated pups during the place preference test. These results indicate that continuous cocaine exposure has selective neurotoxic effects on the habenula of the developing fetus similar to cocaine's effects in the adult.

Diencephalic Neuronal Populations Projecting Axons into the Basal Plate in a Lizard (Gallotia galloti)

Lizard diencephalic populations sending axons into the basal plate were studied by the in vitro HRP technique in the lizard Gallotia. Retrograde labeled cells were concentrated in distinct neuronal groups within alar plates of prosomeres p1 and p3, whereas the alar plate of p2 was poorly labeled. Efferent fibers from alar p1 and p3 populations entered the basal plate of the diencephalon along topologically dorsoventral courses, bifurcating thereafter into longitudinal ascending (rostral) and descending (caudal) trajectories. Thus, diencephalic segments p1 and p3 have alar cell populations contributing to the longitudinal premotor connectivity of the neural axis , whereas the alar p2 segment projects via the fasciculus retroflexus, the efferent tract of the epithalamus. However, the axons from the habenular complex bifurcate within or adjacent to the floor plate and not within the basal plate.

Cyclic AMP phosphodiesterases are localized in regions of the mouse brain associated with reinforcement, movement, and affect

Four cyclic AMP-specific, rolipram-inhibited phosphodiesterases (PDE4s) have been identified in mammals; all four are homologs of dunce, a gene required for learning and memory in Drosophila. To determine the distribution of PDE4s in the mammalian brain, specific antibodies were generated against the proteins encoded by each of three dunce homologs PDE4A, PDE4B, and PDE4D in the mouse. On Western blots, these antibodies recognized multiple protein species in all brain regions studied. Immunohistochemical studies showed that both cell bodies and neuropil were well labeled in selected regions throughout the brain. Immunoreactivity for PDE4A was found predominantly in the anterior olfactory nucleus, subiculum, layer V pyramidal neurons from the cerebral cortex, and corticospinal tracts. By contrast, anti-PDE4B-labeled neurons were observed in the inferior olive, the paraventricular and supraoptic nuclei of the hypothalamus, and in the ventral striatum. Regions of neuropil containing high levels of PDE4B immunoreactivity included the cerebellar molecular layer, globus pallidus, nucleus accumbens, and substantia nigra. Anti-PDE4D antibody distinctly labeled cerebellar Purkinje cells as well as neurons in the medial habenula and thalamic nuclei. Fibers in the fasciculus retroflexus, interpeduncular nuclei, and periaqueductal gray were also stained with this antibody. These findings indicate that the distribution of PDE4s in the brain is remarkably segregated, and suggest that each of these enzymes has a unique functional role. Furthermore, the data support the notion that rolipram, the PDE4-specific inhibitor that acts as an antidepressant in humans, may mediate its behavioral effects through PDE4B, which is highly localized to neural pathways known to underlie reward and affect in mammals.

Subnuclear organization of the rat habenular complexes.

The habenular complexes represent phylogenetically constant structures in the diencephalon of all vertebrates. Available evidence suggests that this area is engaged in a variety of important biological functions, such as reproductive behaviors, central pain processing, nutrition, sleep-wake cycles, stress responses, and learning. Based on Nissl-stained sections, one medial nucleus and two lateral nuclei (divisions) have been widely accepted in the rat. Cytochemical, hodologic, and functional studies suggest a considerably more complex subnuclear structure. To improve our knowledge of the precise structural composition of the habenular complexes, we have systematically investigated their fine ultrastructure in the rat. Based on the detailed analysis of complete series of large, semithin sections supplemented with electron photomicrographs of selected fields, clear criteria for the delineation of five distinct subnuclei of the medial and ten subnuclei of the lateral habenular complexes were elaborated for the first time. All 15 subnuclei were reconstructed, and their dimensions were determined. A medial and lateral stria medullaris were described. Different roots of the fasciculus retroflexus were differentiated within the medial and lateral habenular complexes. The topographical relationships with respect to the adjacent habenular areas as well as to the neighboring thalamic nuclei were identified and demonstrated. The new understanding of the subnuclear organization of the habenular complexes certainly will facilitate further functional investigations. Whether the newly identified subnuclei finally will be recognized as functionally distinct awaits ongoing immunocytochemical, hodologic, and functional studies.

A di-synaptic projection from the superior colliculus to the head of the caudate nucleus via the centromedian-parafascicular complex in the cat: an anterograde and retrograde labeling study

Centromedian-parafascicular (CM-Pf) complex of the thalamus receives inputs from the superior colliculus (SC). The CM-Pf neurons, in turn, project to the neostriatum. In order to establish a circuitry that the efferent terminals of SC neurons make direct synaptic contacts with thalamostriatal neurons in the CM-Pf complex, the anterograde tracer (biocytin) was injected into the unilateral SC and the retrograde tracer (wheat germ agglutinin conjugated to horseradish peroxidase, WGA–HRP) into the ipsilateral head of the caudate nucleus of the cat. The anterogradely labeled SC fibers and their terminals and retrogradely labeled CM-Pf neuronal elements were examined under light and electron microscope. At the light micrsocopic level, biocytin-labeled terminal-like varicosities were observed densely in the latero-dorsal part of the CM and the dorsal part of the Pf lateral to the fasciculus retroflexus. These varicosities were often in close proximity to HRP-labeled somata and dendrites of CM-Pf neurons. Electron microscopic analysis revealed that the biocytin-labeled synaptic boutons contained mainly round synaptic vesicles and established asymmetrical synaptic contacts with retrogradely labeled thalamostriatal neuronal elements, including perikarya, and small and large dendrites. In addition, anterogradely labeled terminals made synaptic contacts with unlabeled somata, small and large dendrites, and spines as well as profiles containing synaptic vesicles. These vesicle-containing profiles were considered to be pre-synaptic elements contacting on thalamostriatal neurons or the vesicle-containing dendrites of local circuit neurons. These results demonstrate that the neurons of the CM-Pf complex of the thalamus is involved in a di-synaptic tecto-striatal circuit.

Cocaine-induced changes in glutamate and GABA immunolabeling within rat habenula and nucleus accumbens.

We previously reported that subchronic administration of cocaine for 5 days via slow-release pellets results in pronounced degeneration in the lateral habenula (LHB) and its primary efferent tract, the fasciculus retroflexus [Ellison (1992): Brain Res 598:353-356; Ellison and Switzer (1993): Neuroreport 5:17-20]. The lateral habenula receives both GABA and glutamate afferents. In order to test the hypothesis that the cocaine-induced degeneration of the fasciculus retroflexus may be related to changes in synaptic activity of either GABA or glutamate nerve terminals within the LHB, the density of nerve terminal immunolabeling of either neurotransmitter was quantified after 5 days of chronic drug administration followed by either 1 or 14 days off the drug. The shell of the nucleus accumbens (NACs) was also analyzed, since this area is thought to be associated with the reward aspects of addictive stimulant drug administration and was previously shown not to be associated with fiber degeneration. We found that cocaine treatment resulted in a significant decrease in the density of nerve-terminal GABA immunolabeling located within the LHB in animals taken off the drug for either 1 or 14 days, while there was no change in the density of glutamate immunolabeling. In the NACs, there was a decrease in the density of glutamate immunolabeling within nerve terminals 1 day but not 14 days after cocaine administration. There was no change in the density of GABA immunolabeling within the NACs following the 1 or 14 day-off period. These results suggest that there are long-term changes in the density of GABA immunolabeling within the LHB and that the effects seen in glutamate synapses within the NACs are transitory. The long-term decrease in GABA immunolabeling within the LHB is consistent with the hypothesis that a decrease in inhibitory synaptic activity, leading to increased excitatory influence on LHB neurons, may result in neurotoxicity and the subsequent degeneration of the fasciculus retroflexus.

The fasciculus retroflexus controls the integrity of REM sleep by supporting the generation of hippocampal theta rhythm and rapid eye movements in rats

The fasciculus retroflexus (FR) fiber bundle comprises the intense cholinergic projection from the medial division of the habenula nucleus (Hbn) of the epithalamus to the interpeduncular nucleus (IPN) of the limbic midbrain. Due to the widespread connections of the Hbn and IPN, it could be surmised that the FR is integrated in the processings of various subsystems that are known to be involved in the sleep–wake mechanisms; relevant sites include the limbic forebrain and midbrain areas and more caudal pontine structures. Consequently, the present study addressed the significance of the FR in the spontaneous sleep–wake stage-associated variations of the different activity patterns of frontal cortex and hippocampal electroencephalograms (EEGs), the electrooculogram, and body movements, in freely behaving rats that had been subjected to either bilateral electrolytic lesioning of the FR or control operations. The evolution of different state combinations was assessed by the combinatory analysis of different activity stages appearing on the 6-h records. As compared to the control-operated group, the FR lesioning substantially reduced the time spent in rapid eye movement (REM) sleep by 79%, moderately decreased the duration of the intermediate state of sleep by 29%, and quiet waking state by 44%, but had virtually no effects on the durations of different types of non-REM sleep (i.e., drowsiness that which involved quiet sleep or slow-wave sleep containing delta and spindle state components) or on the times of active waking behavior that corresponded to the body movements. Quantitative decomposition analyses revealed marked variations in the frontal cortex and hippocampal activity as well as REM during the course of the extracted sleep–wake stages described and there were also some group differences. Of those individual features that were used to determine different sleep–wake stages, the overall hippocampal theta time (41% decrease) and single REM frequency (71% reduction during the REM sleep) were most affected. In contrast, the various properties of desynchronization/synchronization patterns of frontal cortex EEGs were consistently hardly influenced by the FR lesioning. Therefore, the present data suggest the involvement of the FR in the REM sleep processes by establishing prominent associations with the limbic and REM control mechanisms that involve the hippocampus and plausibly pontine ocular activity networks.

Asymmetry in the Left and Right Habenulo-Interpeduncular Tracts in the Frog

In the frog Rana esculenta the left dorsal habenula includes a lateral and a medial component, whereas the right dorsal habenula is only represented by one nucleus. The efferents of the habenular nuclei to the interpeduncular nucleus were herein investigated with the retrograde horseradish peroxidase tracing. Injections of cobaltic–lysine complex in the interpeduncular nucleus were also performed. Intensely labeled fibers of the fasciculus retroflexus on the right and left sides of the brain were found to reach the interpeduncular nucleus from the habenular nuclei running prevalently in two routes—one through the medial, and the other through the lateral region of the diencephalon. On the right side, these fibers originated from the entire dorsal habenula. On the left side, the fibers of the medial route derived from the medial habenular subnucleus, while those of the lateral route derived from the lateral habenular subnucleus. In the dorsal habenulae of both sides, a large number of neurons displayed a Golgi-like labeling, while few such cells were detected in the ventral habenulae. Labeled neurons in the right dorsal habenula resembled those labeled in the lateral subnucleus of the left dorsal habenula, while larger and ramified neurons were detected in the left medial subnucleus. The present findings provide the first description of the pathway originating from the medial and the lateral subnucleus of the left dorsal habenula in the frog and point out that projection neurons of the medial habenular subnucleus are morphologically different from those of the other habenular nuclei. The present data indicate that in the frog the habenular asymmetry could underlie distinct functional correlates of the left and right habenulae.

The Timing and Sequence of Appearance of Neuromeres and Their Derivatives in Staged Human Embryos

Serial sections of 215 human embryos from Carnegie stages 6-17 were investigated, and 85 graphic reconstructions were prepared. It is proposed that neuromeres be defined as morphologically identifiable transverse subdivisions perpendicular to the longitudinal axis of the embryonic brain and extending onto both sides of the body. It is proposed further that primary neuromeres be redefined as the early-appearing larger divisions of the open neural folds, and secondary neuromeres as the smaller subdivisions that are found both before and after closure of the neural tube. In the light of these definitions, 6 primary neuromeres can be detected in the human brain at stage 9, and a maximum of 16 secondary neuromeres at stage 14. The relationships of the 8 rhombomeres to the associated neural crest, as well as to the pharyngeal arches and the exits of the cranial nerves, are tabulated. Rhombomere 8 (Rh. 8) is intermediate between the more rostral neuromeres and the spinal cord, and its neural relationships indicate that the four occipital somitic pairs do not impress a strictly repetitive pattern as in the spinal cord. Hence, it is suggested that Rh. 8 depends on both intrinsic and extrinsic factors. The synencephalon, parencephalon, and isthmic neuromere can be distinguished in stage 13. In stage 14, rostral and caudal portions of the parencephalon are recognizable, and the full complement of 16 neuromeres is now present. The medial ventricular eminence appears in the diencephalon (D1). A longitudinal organisation begins to be superimposed on the neuromeres, as now indicated by the appearance of the hypothalamic cell cord. This continues in stage 15, when the hypothalamic sulcus develops. That groove, however, is not continuous with the sulcus limitans. In the diencephalon, five longitudinal zones can be discerned. In stage 16, fibre tracts, such as the habenulo-interpeduncular (fasciculus retroflexus) and the tract of the posterior commissure, outline the boundaries of the synencephalon. In stage 17, the tract of the zona limitans intrathalamica (along the marginal ridge in the parencephalon) is an important landmark. This is the last stage in which all the neuromeres can be distinguished. The supramamillary recess becomes defined and is the termination of the sulcus limitans: the alar/basal distinction is inappropriate in the human forebrain. The number and identity of the neuromeres in the human brain, their precise sequence of appearance, and the stages at which they appear are here clarified for the first time. The results of various studies of domains of gene expression indicate that, although in some instances such territories follow the morphological neuromeres, in others they may cross interneuromeric boundaries. It is concluded that the precise morphological study of neuromeres in any given species is necessary for correlative investigations of gene expression.

Lesion of the habenular efferent pathway produces anxiety and locomotor hyperactivity in rats: a comparison of the effects of neonatal and adult lesions

Recent studies have implicated the habenula in modulating states of arousal and chronic responses to stress. We examined whether lesion of the habenula efferent pathway, the fasciculus retroflexus (FR), at either 3 (P3) or 70 (P70) days of age affects stress-related anxiety (elevated plus-maze test) and activity levels (open-field test) in rats tested as adults. Both P3- and P70-lesioned rats showed chronically elevated plasma levels of corticosterone. Rats receiving FR lesions as neonates (P3) exhibited greater open arm avoidance on the elevated plus-maze than controls 2 months postoperatively, suggesting a heightened state of anxiety. In contrast, P70-lesioned rats behaved similarly to controls on the plus-maze, but showed increased locomotion and increased grooming in the open field, effects not observed in P3-lesioned rats. When an additional stressful condition was imposed (5 days of social isolation plus 24 h food deprivation) before testing, both FR-lesion groups showed an attenuation of the normal behavioral responses (decreased open-arm entries/time in open arms, increased freezing). The effects of FR lesions on activity and behavioral indices of anxiety may be due to disruption of lateral habenular projections to dopaminergic neurons in the ventral tegmentum and/or projections to regions containing high concentrations of benzodiazepine receptors, the median and dorsal raphe and dorsal periaqueductal gray. Behavioral differences observed as a function of lesion age suggest differential capabilities of P3- and P70-lesioned rats to utilize compensatory mechanisms to correct FR lesion-induced deficits.

Afferent and efferent connections of the habenula in the rainbow trout (Oncorhynchus mykiss): an indocarbocyanine dye (DiI) study.

The habenula is a conserved structure in the brain of vertebrates. With the aim of further understanding of the evolution of the habenular system in vertebrates, we studied the afferent and efferent connections of the habenula of the rainbow trout. Experiments included application of the carbocyanine dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) into the habenula, telencephalon, pineal organ, posterior tubercle, and interpeduncular nucleus (IPN). The results obtained reveal a consistent pattern of habenular connections. Most afferents originate from three nuclei, one extending from the preoptic region to the rostral thalamus (the entopeduncular nucleus), the second located in the region of the hypothalamus-posterior tubercle and consisting of large bipolar cells (tuberculohabenular nucleus), and the third in the preoptic region (preoptic nucleus). A few large neurons of the locus coeruleus appeared to be labeled in some cases. The trout habenula also receives pineal and parapineal projections. Small labeled glial cells were observed in the thalamus around the fasciculus retroflexus and, sometimes, around the IPN. The most conspicuous efferents coursed in the fasciculus retroflexus to the IPN, the isthmal raphe, and the central gray. The existence of olfactohabenular or habenulotelencephalic projections is discussed.

Immunohistochemical localization of μ‐opioid receptors in the central nervous system of the rat

Of the three major types of opioid receptors (μ, δ κ) in the nervous system, μ-opioid receptor shows the highest affinity for morphine that exerts powerful effects on nociceptive, autonomic, and psychological functions. So far, at least two isoforms of μ-opioid receptors have been cloned from rat brain. The present study attempted to examine immunohistochemically the distribution of μ-opioid receptors in the rat central nervous system with two kinds of antibodies to recently cloned μ-opioid receptors (MOR1 and MOR1B). One antibody recognized a specific site for MOR1, and the other bound to a common site for MOR1 and MOR1B. Intense MOR1-like immunoreactivity (LI) was seen in the ‘patch’ areas and subcallosal streak in the striatum, medial habenular nucleus, medial terminal nucleus of the accessory optic tract, interpeduncular nucleus, median raphe nucleus, parabrachial nuclei, locus coeruleus, ambiguus nucleus, nucleus of the solitary tract, and laminae I and II of the medullary and spinal dorsal horns. Many other regions, including the cerebral cortex, amygdala, thalamus, and hypothalamus, also contained many neuronal elements with MOR1-LI. The distribution pattern of the immunoreactivity revealed with the antibody to the common site for MOR1 and MOR1B (MOR1/1B-LI) was almost the same as that of MOR1-L1. Both MOR1-LI and MOR1/1B-LI were primarily located in neuronal cell bodies and dendrites. However, the immunoreactivities were observed in the accessory optic tract, fasciculus retroflexus, solitary tract, and primary afferent fibers in the superficial layers of the medullary and spinal dorsal horns. The presynaptic location of MOR1-LI and MOR1/1B-LI was confirmed by lesion experiments: Enucleation, placing a lesion in the medial habenular nucleus, removal of the nodose ganglion, or dorsal rhizotomy resulted in a clear reduction of the immunoreactivities, respectively, in the nuclei of the accessory optic tract, some subnuclei of the interpeduncular nucleus, nucleus of the solitary tract, or laminae I and II of the spinal dorsal horn. The results indicate that the μ-opioid receptors are widely distributed in the brain and spinal cord, mainly postsynaptically and occasionally presynaptically. Opioids, including morphine, may inhibit the excitation of neurons via the postsynaptic μ-opioid receptors, and also suppress the release of neurotransmitters and/or neuromodulators from axon terminals through the presynaptic μ-opioid receptors. © 1996 Wiley-Liss, Inc.

Immunohistochemical localization of ?-opioid receptors in the central nervous system of the rat

Of the three major types of opioid receptors (μ, δ κ) in the nervous system, μ-opioid receptor shows the highest affinity for morphine that exerts powerful effects on nociceptive, autonomic, and psychological functions. So far, at least two isoforms of μ-opioid receptors have been cloned from rat brain. The present study attempted to examine immunohistochemically the distribution of μ-opioid receptors in the rat central nervous system with two kinds of antibodies to recently cloned μ-opioid receptors (MOR1 and MOR1B). One antibody recognized a specific site for MOR1, and the other bound to a common site for MOR1 and MOR1B. Intense MOR1-like immunoreactivity (LI) was seen in the ‘patch’ areas and subcallosal streak in the striatum, medial habenular nucleus, medial terminal nucleus of the accessory optic tract, interpeduncular nucleus, median raphe nucleus, parabrachial nuclei, locus coeruleus, ambiguus nucleus, nucleus of the solitary tract, and laminae I and II of the medullary and spinal dorsal horns. Many other regions, including the cerebral cortex, amygdala, thalamus, and hypothalamus, also contained many neuronal elements with MOR1-LI. The distribution pattern of the immunoreactivity revealed with the antibody to the common site for MOR1 and MOR1B (MOR1/1B-LI) was almost the same as that of MOR1-L1. Both MOR1-LI and MOR1/1B-LI were primarily located in neuronal cell bodies and dendrites. However, the immunoreactivities were observed in the accessory optic tract, fasciculus retroflexus, solitary tract, and primary afferent fibers in the superficial layers of the medullary and spinal dorsal horns. The presynaptic location of MOR1-LI and MOR1/1B-LI was confirmed by lesion experiments: Enucleation, placing a lesion in the medial habenular nucleus, removal of the nodose ganglion, or dorsal rhizotomy resulted in a clear reduction of the immunoreactivities, respectively, in the nuclei of the accessory optic tract, some subnuclei of the interpeduncular nucleus, nucleus of the solitary tract, or laminae I and II of the spinal dorsal horn. The results indicate that the μ-opioid receptors are widely distributed in the brain and spinal cord, mainly postsynaptically and occasionally presynaptically. Opioids, including morphine, may inhibit the excitation of neurons via the postsynaptic μ-opioid receptors, and also suppress the release of neurotransmitters and/or neuromodulators from axon terminals through the presynaptic μ-opioid receptors. © 1996 Wiley-Liss, Inc.

Telencephalo-habenulo-interpeduncular connections in the goldfish: a DiI study.

The telencephalo-habenulo-interpeduncular system has been anatomically and neurochemically characterized in mammals. However, little is known about these important forebrain-midbrain connections in non-mammalian vertebrates, although available data suggest that they are evolutionarily relatively conservative. Previous ultrastructural studies in the goldfish confirmed the presence of massive telencephalohabenular and habenulointerpeduncular projections and demonstrated a minor direct telencephalointerpeduncular connection. Here we report the anterograde and retrograde transport of lipophilic fluorescent carbocyanine dye from the interpeduncular nucleus and the habenular nuclei in the fixed goldfish brain. The application of dye into the interpeduncular nucleus resulted in massive labeling of the fasciculus retroflexus and of the habenular neurons. A few scattered neurons were also seen in the dorsal nucleus of area ventralis telencephali. Application of dye into the habenulae resulted in anterograde transport through the medial and lateral olfactory tracts to some cell bodies in the anterior and posterior zone of area ventralis telencephali and in perikarya of the bed nucleus and in the entopeduncular nucleus. These results demonstrate the origin of the direct telencephalointerpeduncular projection in the goldfish and confirm some important homologies with forebrain-midbrain projections in land vertebrates.

Immunohistochemical localization of the cloned μ opioid receptor in the rat CNS

Three opioid receptor types have recently been cloned that correspond to the pharmacologically defined μ, δ and κ 1 receptors. In situ hybridization studies suggest that the opioid receptor mRNAs that encode these receptors have distinct distributions in the central nervous system that correlate well with their known functions. In the present study polyclonal antibodies were generated to the C terminal 63 amino acids of the cloned μ receptor (335–398) to examine the distribution of the μ receptor-like protein with immunohistochemical techniques. μ receptor-like immunoreactivity is widely distributed in the rat central nervous system with immunoreactive fibers and/or perikarya in such regions as the neocortex, the striatal patches and subcallosal streak, nucleus accumbens, lateral and medial septum, endopiriform nucleus, globus pallidus and ventral pallidum, amygdala, hippocampus, presubiculum, thalamic and hypothalamic nuclei, superior and inferior colliculi, central grey, substantia nigra, ventral tegmental area, interpeduncular nucleus, medial terminal nucleus of the accessory optic tract, raphe nuclei, nucleus of the solitary tract, spinal trigeminal nucleus, dorsal motor nucleus of vagus, the spinal cord and dorsal root ganglia. In addition, two major neuronal pathways, the fasciculus retroflexus and the stria terminalis, exhibit densely stained axonal fibers. While this distribution is in excellent agreement with the known μ receptor binding localization, a few regions, such as neocortex and cingulate cortex, basolateral amygdala, medial geniculate nucleus and the medial preoptic area fail to show a good correspondence. Several explanations are provided to interpret these results, and the anatomical and functional implications of these findings are discussed.

Trans-synaptic modulation of striatal ACh release in vivo by the parafascicular thalamic nucleus.

Electrical stimulation of the parafascicular but not the ventrolateral or dorsomedial thalamic nucleus (ten 0.5 ms, 10 V pulses, 140 microA) of freely moving rats induced a frequency-dependent (2.5, 5, 10 and 20 Hz) increase in the extracellular acetylcholine (ACh) content of the dorsal striatum, assessed by trans-striatal microdialysis. The time-dependent effect of 10 Hz stimulation was studied. The peak increase, 39% above baseline, was attained during 4 min of stimulation. This was blocked by coperfusion with 5 microM tetrodotoxin, indicating that the release we measured represents a physiological process. The facilitatory effect of parafascicular nucleus stimulation does not appear to be associated with indirect action through the cerebral frontal cortex because acute lesion of the excitatory corticostriatal afferents, which by itself reduced basal ACh release by 40%, did not modify the effect of 10 Hz stimulation. The possible involvement of the fasciculus retroflexus in the facilitation of ACh release was also ruled out. The non-competitive NMDA-type receptor antagonist MK-801, applied by reversed dialysis (30 microM) or systemically injected (0.2 mg/kg), significantly reduced the basal ACh output and prevented the tetanus-evoked increase in ACh release. The results provide in vivo evidence that the activity of the cholinergic neurons in the dorsal striatum is trans-synaptically modulated by parafascicular nucleus excitatory afferents through activation of the NMDA subtype of glutamate receptors that is probably located in the striatum.

Dissociation of behavioral changes in rats resulting from lesions of the habenula versus fasciculus retroflexus and their possible anatomical substrates.

Dissociation of behavioral changes in rats resulting from lesions of the habenula versus fasciculus retroflexus and their possible anatomical substrates.