Enkephalinergic innervation of GABAergic neurons in the dorsal raphe nucleus of the rat

The preembedding double immunoreaction method was used to study interrelations of enkephalinergic and GABAergic neuronal elements in the dorsal raphe nucleus of the Wistar albino rat. The enkephalin-like neuronal elements were immunoreacted by the peroxidase-antiperoxidase method and silver-gold intensified, which showed strongly and was specific. The GABA-like immunoreactive neurons were immunoreacted by the peroxidase-antiperoxidase method only. GABA-like neural somata were postsynaptic to both the enkephalin-like immunoreactive and the non-immunoreactive axon terminals. The enkephalin-like immunoreactive axon terminals were also found to synapse GABA-like immunoreactive dendrites. The GABA-like immunoreactive neuronal elements were also found to receive synapses from other non-immunoreactive as well as GABA-like immunoreactive axon terminals. Almost all of the synapses appeared to be asymmetrical. Possible functional activity of interactions among the enkephalinergic, GABAergic, and serotonergic neuronal elements in the dorsal raphe nucleus are discussed.     

Spare receptors and intrinsic activity: studies with D1 dopamine receptor agonists

Anti-nitric oxide synthase antibody was used to study the distribution, cytoarchitecture, and synaptic relations of nitric oxide synthase-like immunoreactive neurons in the whole rostral-caudal length of the dorsal raphe nucleus of the rat and compared them with serotonergic neurons. Results showed that the distribution of the nitric oxide synthase in the dorsal raphe nucleus was similar to that of the serotonergic neurons at the rostral part of the dorsal raphe nucleus, including the mediodorsal and the medioventral cell groups, and changed at the middle and caudal parts of the dorsal raphe nucleus. The cytoarchitecture of the nitric oxide synthase-like immunoreactive neurons in the medioventral cell group of the dorsal raphe nucleus was similar to that of the serotonergic neurons. Similar to the serotonergic neurons there, nitric oxide synthase-like immunoreactive neurons also received synapses from axon terminals that contained round, or flattened vesicles, or both kinds. Different to the serotonergic neurons, the few nitric oxide synthase-like immunoreactive axon terminals that were in this area formed synapses.     

Substance P and enkephalin immunoreactivities in axonal boutons presynaptic to physiologically identified dorsal horn neurons. An ultrastructural multiple-labelling study in the cat

A combination of intracellular electrophysiological recording and injection of horseradish peroxidase with ultrastructural immunocytochemistry was used to investigate the synaptic interplay between substance P- and enkephalin-immunoreactive axonal boutons and three types of functionally characterized dorsal horn neurons in the cat spinal cord. The dorsal horn neurons were classified as nociceptive specific, wide dynamic range and non-nociceptive based on their responses to innocuous and noxious stimuli. Most of the nociceptive neurons (either nociceptive specific or wide dynamic range) contained enkephalin immunoreactivity, but none of the non-nociceptive neurons were positive for enkephalin. Three types of immunoreactive boutons were found in contact with the functionally characterized dorsal horn neurons. These boutons were positive for either substance P, enkephalin, or substance P+enkephalin. Quantitative analysis revealed that the percentages of substance P-immunoreactive boutons apposed to the cell bodies, proximal dendrites and distal dendrites of nociceptive neurons were significantly higher than those of non-nociceptive neurons. Furthermore, the percentages of substance P+enkephalin-immunoreactive axonal boutons apposed to the distal dendrites of nociceptive neurons were significantly higher than those of non-nociceptive neurons and the percentages of enkephalin-immunoreactive boutons apposed to the cell bodies and proximal dendrites of nociceptive neurons were significantly higher than in non-nociceptive neurons. Finally, neither enkephalin-immunoreactive nor substance P+enkephalin-immunoreactive boutons were ever seen presynaptic to substance P-immunoreactive boutons. These results provide evidence of an anatomical substrate within the dorsal horn for the interaction of substance P-mediated with enkephalin-mediated mechanisms. The data support the idea that the modulation of nociceptive input in the dorsal horn by enkephalinergic neurons occurs mainly via a postsynaptic mechanism, and thus suggest that dorsal horn enkephalinergic neurons participate in a local inhibitory feedback loop in a distinct pathway from the previously postulated opioid-mediated depression of substance P release from primary afferent terminals.     

Synaptic relations of neurotensinergic neurons in the dorsal raphe nucleus

The ultrastructure and synaptic relations of neurotensinergic neurons in the rat dorsal raphe nucleus (DRN) were examined. The neurotensin-like immunoreactive (NT-L1) neurons in the DRN were fusiform or spherical. The NT-LI perikarya could only be detected in colchicine-treated animals whereas the immunoreactive axon terminals could only be found in the animals not treated with colchicine. Although many NT-LI dendrites received synapses from nonimmunoreactive axon terminals, the NT-LI perikarya received few synapses. NT-LI axon terminals also made synapses on nonimmunoreactive dendrites. Occasionally, synapses were found between the NT-LI axon terminals and NT-LI dendrites in the cases in which the animals were not treated with colchicine.     

Dopamine neurons make glutamatergic synapses in vitro

Interactions between dopamine and glutamate play prominent roles in memory, addiction, and schizophrenia. Several lines of evidence have suggested that the ventral midbrain dopamine neurons that give rise to the major CNS dopaminergic projections may also be glutamatergic. To examine this possibility, we double immunostained ventral midbrain sections from rat and monkey for the dopamine-synthetic enzyme tyrosine hydroxylase and for glutamate; we found that most dopamine neurons immunostained for glutamate, both in rat and monkey. We then used postnatal cell culture to examine individual dopamine neurons. Again, most dopamine neurons immunostained for glutamate; they were also immunoreactive for phosphate-activated glutaminase, the major source of neurotransmitter glutamate. Inhibition of glutaminase reduced glutamate staining. In single-cell microculture, dopamine neurons gave rise to varicosities immunoreactive for both tyrosine hydroxylase and glutamate and others immunoreactive mainly for glutamate, which were found near the cell body. At the ultrastructural level, dopamine neurons formed occasional dopaminergic varicosities with symmetric synaptic specializations, but they more commonly formed nondopaminergic varicosities with asymmetric synaptic specializations. Stimulation of individual dopamine neurons evoked a fast glutamatergic autaptic EPSC that showed presynaptic inhibition caused by concomitant dopamine release. Thus, dopamine neurons may exert rapid synaptic actions via their glutamatergic synapses and slower modulatory actions via their dopaminergic synapses. Together with evidence for glutamate cotransmission in serotonergic raphe neurons and noradrenergic locus coeruleus neurons, the present results suggest that glutamatergic cotransmission may be the rule for central monoaminergic neurons.     

Localization of tyrosine hydroxylase in neuronal targets and efferents of the area postrema in the nucleus tractus solitarii of the rat

Catecholamines in the nucleus tractus solitarii (NTS) have been implicated in autonomic responses to circulating hormones that act on neurons in the area postrema, the most caudal circumventricular organ in brain. We combined immunoperoxidase labeling of the anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHAL) with immunogold-silver labeling of tyrosine hydroxylase to determine whether this enzymatic marker for catecholamines was present in efferents from the area postrema or their targets in the rat NTS. At survival periods of 10-12 days after PHAL injections into the area postrema, light microscopy revealed numerous varicose processes containing peroxidase reaction product for PHAL in the dorsomedial, medial, and commissural NTS. Some of these labeled processes were located near neuronal perikarya and processes containing immunogold-silver intensified reaction product for tyrosine hydroxylase. Electron microscopy of the commissural and dorsomedial NTS established that the majority of the labeling for PHAL was in axon terminals, whereas immunogold labeling for tyrosine hydroxylase was mainly in soma and dendrites. Only 3 out of 579 PHAL-labeled terminals also contained detectable tyrosine hydroxylase immunoreactivity. Fifty-eight percent (335/579) of the PHAL-labeled terminals formed synapses with recognized symmetric junctions, whereas the remainder lacked synaptic specializations within the examined series of serial sections. Of those PHAL terminals forming recognized symmetric junctions, 22% were on tyrosine hydroxylase-immunoreactive dendrites, 74% on unlabeled dendrites and 4% on unlabeled axon terminals. From a total of 1,250 observed contacts on tyrosine hydroxylase labeled dendrites, 88 (7%) contained PHAL, 9 (< 1%) contained TH, and 1,180 (93%) lacked detectable immunoreactivity and formed primarily symmetric synapses. We conclude that a few catecholamine, but mainly noncatecholamine efferents from the area postrema provide a monosynaptic, and most likely inhibitory input to target neurons both with and without tyrosine hydroxylase immunoreactivity in the dorsomedial and commissural NTS. Synapses between the efferent terminals from the area postrema and tyrosine hydroxylase labeled and unlabeled dendrites as well as unlabeled axons in these specific subnuclei of the NTS suggest multiple sites for modulation of gastric and cardiovascular reflexes in response to circulating peptides.     

Involvement of the area postrema in the regulation of sympathetic outflow to the cardiovascular system

The circumventricular organs of the brain have been implicated in the central regulation of the cardiovascular system. The area postrema, which is the only circumventricular organ in the hindbrain, has received less attention than the others, but recent studies suggest that it may play an important role in the regulation of the cardiovascular system. Studies in rats and rabbits indicate that angiotensin II (Ang II)-dependent hypertension is abolished by lesioning of the area postrema. Additional studies indicate that the hypertension associated with Ang II involves a resetting of the arterial baroreflex to a higher pressure. This upward resetting requires an interaction of neurons in the area postrema with barosensitive neurons terminating in the medial nucleus tractus solitarius (mNTS). Another peptide, arginine vasopressin (AVP), has been shown to enhance the sympathoinhibitory influence of the arterial baroreflex via an action at the area postrema. Studies in rabbits suggest that the sympathoinhibitory response is due to resetting of the baroreflex to a lower pressure. Electrophysiological studies, using an in vitro brain slice preparation, have shown that activation of area postrema neurons projecting to the mNTS alters the responsiveness of mNTS neurons to afferent inputs. It is postulated that alpha-adrenergic mechanisms are involved in these interactions.     

Preproenkephalin messenger RNA-expressing neurons in the rat parabrachial nucleus: subnuclear organization and projections to the intralaminar thalamus

The pontine parabrachial nucleus, which is a key structure in the central processing of autonomic, nociceptive and gustatory information, is rich in a variety of neuropeptides. In this study we have analysed the distribution of parabrachial neurons that express preproenkephalin messenger RNA, which encodes for the precursor protein for enkephalin opioids. Using an in situ hybridization method, we found that preproenkephalin messenger RNA-expressing neurons were present in large numbers in four major areas of the parabrachial nucleus: the K?lliker-Fuse nucleus, the external lateral subnucleus, the ventral lateral subnucleus, and in and near the internal lateral subnucleus. Many preproenkephalin messenger RNA-expressing neurons were also seen in the central lateral subnucleus, and in the medial and external medial subnuclei. Few labeled neurons were found in the dorsal and superior lateral subnuclei. Injection of the retrograde tracer substance cholera toxin subunit B into the midline and intralaminar thalamus demonstrated that the enkephalinergic neurons in and near the internal lateral subnucleus were thalamic-projecting neurons. Taken together with the results of previous tract-tracing studies, the present findings show that many of the enkephalinergic cell groups in the parabrachial nucleus are located within the terminal zones of the ascending projections that originate from nociresponsive neurons in the medullary dorsal horn and spinal cord, as well as from viscerosensory neurons within the nucleus of the solitary tract. The enkephalinergic neurons in the parabrachial nucleus may thus transmit noci- and visceroceptive-related information to their efferent targets. On the basis of the present and previous observations, we conclude that these targets include the intralaminar and midline thalamus, the ventrolateral medulla and the spinal cord. Through these connections, nociceptive and visceroceptive stimuli may influence several functions, such as arousal, respiration and antinociception.     

Co-localization of serotonin and FMRFamide-like immunoreactivities in the central nervous system of the earthworm, Eisenia fetida

In addition to receptor-type pinealocytes, the mammalian pineal organ contains small and large neurons and ependymal/glial cells as well. Axons of pinealocytes form synaptic ribbon-containing axo-dendritic synapses on large secondary pineal neurons and/or terminate as neurohormonal endings on the basal lamina of the vascular surface of the organ. The small pineal neurons were found to be gamma-aminobutyric acid (GABA)-immunoreactive, while large secondary neurons and pinealocytes contained immunoreactive amino acids (glutamate and aspartate). Glutamate accumulated presynaptically in pinealocytic axon terminals on large secondary neurons and in the axons of these neurons. Glutamate immunoreactive axons of pineal neurons were traced via the pineal tract to the habenular nucleus. Axons containing granular vesicles and coming from extrapineal perikarya are glutamate immunoreactive as well. Aspartate and GABA are also present in some of the myelinated axons, supposedly pinealopetal in the pineal tract.     

Substance P nerve terminals synapse upon negative chronotropic vagal motoneurons

Previous data indicate that there are anatomically segregated and physiologically independent parasympathetic ganglia on the surface of the heart which are capable of selective control of sino-atrial rate, atrio-ventricular conduction, and atrial contractility. We have injected a retrograde tracer into the cardiac ganglion which selectively regulates heart rate (the SA ganglion). Medullary tissues were processed for the histochemical visualization of retrogradely labeled neurons and for the immunohistochemical detection of the neurotransmitter substance P (SP) by dual labeling light and electron microscopic methods. Negative chronotropic retrogradely labeled cells were found in a long slender column in the ventrolateral nucleus ambiguous (NA-VL) which enlarged somewhat at the level of the area postrema. These cells were found bilaterally, but they were asymmetrically distributed. Half the animals showed a pronounced right side predominance in retrograde labeling, while the other half of the animals showed a lesser left side predominance. These observations may help to explain some of the controversy in the literature concerning the relative influence of the right and left vagus nerves on sinus rate. Ultrastructural examination demonstrated axo-somatic and axo-dendritic contacts between SP nerve terminals and retrogradely labeled negative chronotropic NA-VL neurons. SP immunoreactivity was often associated with large dense-core vesicles in terminals forming either symmetric or asymmetric synapses. These observations provide a potential anatomical substrate for the centrally mediated bradycardia elicited by microinjections of SP into the NA. SP immunoreactive terminals were also observed to make axo-somatic, axo-dendritic, and axo-axonic synapses with unlabeled neurons in NA-VL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Noradrenergic input to nociceptive modulatory neurons in the rat rostral ventromedial medulla

Within the rostral ventromedial medulla (RVM), there are two classes of putative pain modulation neurons: ON cells and OFF cells, which respectively burst or pause prior to withdrawal reflexes elicited by noxious stimulation. Alpha-adrenergic agonists injected into the RVM produce changes in the latency of spinal nocifensive reflexes and, when iontophoretically applied, alter the firing of RVM ON but not OFF cells. To provide further information about the contribution of norepinephrine to RVM neuron function, we analyzed the distribution of tyrosine hydroxylase immunoreactive (TH-ir) appositions upon RVM ON and OFF cells. In the lightly anesthetized rat, seven ON and five OFF cells were identified by changes in their discharge rate in relation to nociceptive withdrawal reflexes and were labeled by intracellular injection of neurobiotin. Sections containing labeled cells were visualized by using avidin conjugated to a Texas Red fluorophore. Tissue with labeled cells was subsequently processed for TH-ir by using a Bodipy fluorophore conjugated secondary antibody. The distribution of the Bodipy-labeled fibers and terminals upon the Texas Red-labeled neurons was mapped using a confocal laser-scanning microscope. All the labeled neurons exhibited close TH-ir appositions. Appositions were of two types: swellings and fibers. Although the numbers and density of appositions varied among the cells, there were no consistent differences that correlated with physiological properties. Thus the overall density of appositions for ON cells (29.0 +/- 22.2 x 10(4) microns2) did not differ significantly from that for OFF cells (25.4 +/- 22.2 x 10(4) microns2). Tyrosine hydroxylase immunoreactive (TH-ir) appositions upon ON and OFF cells varied with their location along the dorso-ventral axis with more ventral neurons having a greater density of TH-ir swelling-type appositions. In a separate study, TH-ir and dopamine-beta-hydroxylase-like immunoreactivity (DBH-ir) were mapped in the same sections by using confocal microscopy. Nearly 97% of the TH-ir profiles co-localized with DBH-ir. These observations provide evidence that both ON and OFF cells in the RVM are targeted by noradrenergic inputs.     

Catecholaminergic systems in the zebrafish. III. Organization and projection pattern of medullary dopaminergic and noradrenergic neurons

To characterize the catecholaminergic systems in the zebrafish medulla, immunocytological studies were performed by using antibodies directed against tyrosine hydroxylase and dopamine-beta-hydroxylase. Catecholaminergic neurons could be categorized into three populations based on location, dendritic morphology, axonal projection pattern, and targets: an interfascicular group, a vagal area group, and an area postrema group. All groups contained both dopaminergic and noradrenergic neurons. Interfascicular neurons formed a loose longitudinal column of approximately 16-20 multipolar neurons on each side of the medulla, whose rostrocaudal extension coincided roughly with the vagal lobe. These neurons were relatively large and had dendrites that arborized throughout the reticular formation and in the vagal lobe. They also contributed axonal processes to the longitudinal catecholamine bundle. Neurons associated with structures in the vagal area were mostly dopaminergic. Some cells had a short, thin apical process that arborized into a dense plexus near the ventricular surface, and all cells had a basal dendrite that divided into two main branches: one extended caudally to terminate in the commissural nucleus of Cajal and among the postobecular catecholaminergic cell group; the other extended laterally and joined the longitudinal catecholamine bundle. The caudal extent of this cell group reached the medullospinal junction. The area postrema cell group consisted of densely packed, bipolar neurons. One process of these neurons contacted the ventricular surface in the area postrema, and one terminated in the commissural nucleus of Cajal. Collaterals from the latter innervated the superficial laminae of the vagal lobe and joined the longitudinal catecholamine bundle. The longitudinal catecholamine bundle ascended through the medulla ventral to the secondary gustatory tract. Whether some fibers extended more rostrally is not known. The majority of the terminal fields of medullary catecholaminergic neurons appeared to be restricted to the medulla and were strongly associated with sensory systems. With the exception of some cells in the vagal area, catecholamine-containing neurons in the zebrafish medulla were not obviously homologous to those in the mammalian brainstem.     

Identification and localization of an immunoreactive AMPA-type glutamate receptor subunit (GluR4) with respect to identified photoreceptor synapses in the outer plexiform layer of goldfish retina

L-glutamate, the main excitatory synaptic transmitter in the retina, is released from photoreceptors and evokes responses in second-order retinal neurons (horizontal, bipolar cells) which utilize both ionotropic and metabotropic types of glutamate receptors. In the present study, to elucidate the functional roles of glutamate receptors in synaptic transmission, we have identified a specific ionotropic receptor subunit (GluR4) and determined its localization with respect to photoreceptor cells in the outer plexiform layer of the goldfish retina by light and pre-embedding electron-microscopical immunocytochemistry. We screened antisera to mammalian AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate)-preferring ionotropic glutamate receptors (GluR 1-4) of goldfish retina by light- and electron-microscopical immunocytochemistry. Only immunoreactive (IR) GluR4 was found in discrete clusters in the outer plexiform layer. The cones contacted in this manner were identified as long-wavelength ("red") and intermediate-wavelength ("green") cones, which were strongly immunoreactive to monoclonal antibody FRet 43 and antisera to goldfish red and green-cone opsins; and short-wavelength ("blue") cones, which were weakly immunoreactive to FRet 43 but strongly immunoreactive with antiserum to blue-cone opsin. Immunoblots of goldfish retinal homogenate with anti-GluR4 revealed a single protein at M(r) = 110 kDa. Preadsorption of GluR4 antiserum with either the immunizing rat peptide, or its goldfish homolog, reduced or abolished staining in retinal sections and blots. Therefore, we have detected and localized genuine goldfish GluR4 in the outer plexiform layer of the goldfish retina. We characterized contacts between photoreceptor cells and GluR4-IR second-order neurons in the electron microscope. IR-GluR4 was localized to invaginating central dendrites of triads in ribbon synapses of red cones, semi-invaginating dendrites in other cones and rods, and dendrites making wide-cleft basal junctions in rods and cones; the GluR4-IR structures are best identified as dendrites of OFF-bipolar cells. The results of our studies indicate that in goldfish retina GluR4-expressing neurons are postsynaptic to all types of photoreceptors and that transmission from photoreceptors to OFF-bipolars is mediated at least in part by AMPA-sensitive receptors containing GluR4 subunits.     

Apposition of enkephalin- and neurotensin-immunoreactive neurons by serotonin-immunoreactive varicosities in the rat spinal cord

The descending serotonergic system provides a powerful inhibitory input to the dorsal horn of the spinal cord. Little is known about the chemical identity of the spinal neurons that the serotonergic system innervates, although spinal enkephalinergic neurons are likely candidates. This study investigated the apposition of serotonin-immunoreactive varicosities onto enkephalin- and neurotensin-immunoreactive neurons in the rat lumbosacral spinal cord. Using a double immunofluorescence technique, serotonin-immunoreactive varicosities were observed to abut the soma or proximal dendrites of [Met]enkephalin- and neurotensin-immunoreactive neurons. Nearly 75% of all [Met]enkephalin- and neurotensin-immunoreactive neurons were apposed by serotonin-immunoreactive varicosities in the marginal zone and dorsal gray commissure. In substantia gelatinosa, approximately half of the [Met]enkephalin- and neurotensin-immunoreactive neurons were juxtaposed by serotonin-immunoreactive varicosities. [Met]enkephalin-immunoreactive neurons also were bordered by serotonin-immunoreactive varicosities in the nucleus proprius (65%) and sacral parasympathetic nucleus (75%). The results of this study suggest that the descending serotonergic system mediates nociception via probable contacts with intrinsic enkephalin and neurotensin spinal systems. The mode of action of spinal serotonin on enkephalin and neurotensin neurons may be through "volume" transmission vs synaptic or "wiring" transmission.     

Patterns of excitatory and inhibitory synaptic transmission in the rat neostriatum as revealed by 4-AP

1. Synaptic potentials induced by 4-aminopyridine (4-AP) were recorded intracellularly from rat neostriatal neurons in an in vitro slice preparation. EC50 for this 4-AP action was approximately 120 microM. The threshold for activation of synaptic potentials was 5 microM. 2. 4-AP-induced synaptic potentials appeared stochastically. Most were blocked by 1 microM tetrodotoxin or 400 microM Cd2+. Therefore they reflect a release of neurotransmitters dependent on both Ca2+ entry to the terminals and action potential firing. 3. Bicuculline (BIC) (< or = 10 microM), a gamma-aminobuturic acid-A (GABAA) antagonist, blocked about half of the 4-AP-induced synaptic potentials. This suggests that intrinsic inhibitory connections within the neostriatum are activated by 4-AP administration. 4. 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; < or = 10 microM) plus D-2-amino-5-phosphonovaleric acid (D-APV; < or = 100 microM) blocked most of the BIC-resistant 4-AP-induced synaptic potentials. This suggests that 4-AP induced release of glutamate (GLU) from extrinsic glutamatergic afferents. As most glutamatergic afferents are extrinsic, these afferents then would be able to fire spikes and release transmitter for several hours after they are cut from their somata. 5. If CNQX plus D-APV were administered before BIC, neostriatal neurons responded in different ways. In one half of the neurons, all induced synaptic potentials were blocked. This suggests that most GABAergic intrinsic connections between neostriatal neurons are activated indirectly by 4-AP. 4-AP would first activate extrinsic glutamatergic afferents and these in turn would activate GABAergic intrinsic neurons and connections. 6. In the remaining half of the recorded neurons, administration of CNQX plus D-APV blocked most, but not all of the 4-AP-induced synaptic potentials. The synaptic potentials that remained had a characteristic pattern: they were high amplitude, rhythmic, bursts of synaptic potentials. They were blocked by BIC (5 microM) but not by mecamylamine (> 10 microM). This suggests that these bursts of synaptic potentials were GABAergic and generated by intrinsic neurons. Therefore these neurons would not innervate all neostriatal neurons equally but just a subset of them. 7. Records from an identified aspiny neostriatal interneuron, obtained from the same preparation, are shown. This interneuron fired in bursts and its morphologically and physiologically similar to the recently described, fast spiking, parvalbumin immunoreactive, GABAergic, aspiny interneuron is functional in the slice preparation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Tyrosine hydroxylase-like immunoreactivity in the brain of the teleost fish Tinca tinca

The distribution of tyrosine hydroxylase-like immunoreactivity has been studied in the central nervous system of the tench (Tinca tinca) using a monoclonal antibody and the avidin-biotin-immunoperoxidase technique. Immunoreactive elements were found in all brain subdivisions. Thus, catecholaminergic neurons and fibers were detected in most nuclei of the ventral telencephalon and in the pars centralis and lateralis of the dorsal telencephalon. The diencephalon was the brain subdivision where largest density of immunoreactive elements were found, mainly located in the periventricular region. The mesencephalon and metencephalon only demonstrated immunoreactive fibers, and no immunoreactive cell bodies were observed in these regions. The myelencephalon showed three groups of immunoreactive neurons located at isthmal level, in the central medullary area, and at the medullary-spinal cord transition area. The distribution of catecholaminergic elements in the tench brain revealed a general pattern shared by most teleosts. The number and distribution of catecholaminergic elements was similar to those described in other teleostean species in the caudal region of the brain. However, noticeable differences were found in areas related to the integration of different sensory information, specially in the telencephalon and diencephalon, suggesting a relationship among the functional level of each sensorial system and the complexity of the catecholaminergic innervation of their integration regions. Additionally, this study revealed the presence of an important number of cerebrospinal fluid-contacting cells in the organum paraventricularis expressing tyrosine hydroxylase that in most investigated teleostean species were tyrosine hydroxylase-immunonegative despite they contained catecholamines. This data argues for distinct evolutionary patterns in the hypothalamic catecholaminergic system among different teleostean species.     

GABA(A) receptors in the primate basal ganglia: an autoradiographic and a light and electron microscopic immunohistochemical study of the alpha1 and beta2,3 subunits in the baboon brain

The distribution of gamma-aminobutyric acid(A) (GABA(A)) receptors was investigated in the basal ganglia in the baboon brain by using receptor autoradiography and the immunohistochemical localisation of the alpha1 and beta2,3 subunits of the GABA(A) receptor by light and electron microscopy. In the caudate-putamen, the alpha1 subunit was distributed in high densities in the matrix compartment, and the beta2,3 subunits were more homogeneously distributed; the globus pallidus showed lower levels of the alpha1 and beta2,3 subunits. Four types of alpha1 subunit immunoreactive neurons were identified in the baboon striatum: the most numerous (75%) were type 1 medium-sized aspiny neurons; type 2 (2%) were large aspiny neurons with an indented nuclear membrane located in the ventral striatum; type 3 neurons were the least numerous (1%) and were comprised of large neurons in the ventromedial regions of the striatum; and type 4 (22%) neurons were medium to large aspiny neurons located in striosomes. At the ultrastructural level, alpha1 and beta2,3 subunit immunoreactivity was localised in the neuropil of the striatum in both symmetrical and asymmetrical synaptic contacts. In the globus pallidus, alpha1 and beta2,3 subunits were localised on large neurons and were found in three types of synaptic terminals: type 1 terminals were small and established symmetrical synapses; type 2 terminals were large; and type 3 terminals formed small synaptic terminals with subjunctional dense bodies. These results show that the subunit composition of GABA(A) receptors varies between the striosome and the matrix compartments in the striatum and that there is receptor subunit homogeneity in the globus pallidus.     

Novel associations among gonadotropin-releasing hormone neurons

GnRH is secreted in bursts into the hypophyseal portal vasculature by a small dispersed population of neurons. The means by which the activity of these intrinsically pulsatile cells is coordinated are unknown. This study was initiated as a continuation of our examination of the synaptic input to these cells and their anatomical relationships. Brain tissue from female rhesus monkeys and male and female rats was prepared for the immunocytochemical demonstration of GnRH. At the light microscopic level, GnRH neurons were occasionally found to be in close apposition. Such pairs (or small groups) were randomly distributed throughout the population of GnRH neurons from the diagonal band of Broca through the anterior hypothalamic area in rats and monkeys and in the medial basal hypothalamus in monkeys. The percentage of neurons found in such associations was small (2-7% in rats and 3-15% in monkeys) and was independent of the hormonal condition of the animal. GnRH neurons, either singly or in pairs, were serially sectioned for electron microscopic examination. The sparsity of synaptic input to the cell body that we had reported earlier on the basis of random sampling was confirmed. No soma had more than a dozen synapses, but none totally lacked innervation. The most significant result of serial reconstruction was the discovery of intercellular bridges or passageways between contiguous pairs of GnRH neurons. These were formed by the fusion of processes extending from the two cells or by fusion and opening of passageways in the membranes along regions of contiguity between the two cells. They were found in four of seven pairs of neurons examined in the rat and in four of eight pairs in the monkey. This syncytial arrangement along with GnRH-GnRH synaptic interactions could contribute to the coordination of dispersed influences on these neurons and the propagation of coordinated pulsatile release of GnRH.     

Modulation of quinolinic acid-induced depletion of striatal NADPH diaphorase and enkephalinergic neurons by inhibition of nitric oxide synthase

The present study was designed to examine the role of nitric oxide (NO) in quinolinic acid (QUIN)-induced depletion of rat striatal nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase and enkephalinergic neurons. Intrastriatal injection of QUIN produced a dose-dependent decrease in NADPH diaphorase and enkephalin positive cells, with cell loss being evident following the injection of 6 and 18 nmol QUIN, respectively. To evaluate the role of NO in QUIN-induced toxicity, animals were pretreated with the non-specific nitric oxide synthase (NOS) inhibitor, Nomega-nitro-l-arginine (l-NAME) or the selective neuronal NOS inhibitor, 7-nitro indazole (7-NI). l-NAME (2x250 mg/kg, i.p. 8 h apart) maximally inhibited striatal NOS activity by 85%, while 7-NI (50 mg/kg, i.p.) maximally inhibited striatal NOS activity by 60%. Pretreatment with l-NAME or 7-NI potentiated the loss of NADPH diaphorase neurons resulting from intrastriatal injection of low doses of QUIN (18 nmol). Neither NOS inhibitor had any effect on the loss of striatal NADPH diaphorase neurons induced by a higher dose of QUIN (24 nmol). In contrast, 7-NI partially prevented the QUIN (18 and 24 nmol)-induced loss of enkephalinergic neurons, while l-NAME had no effect. These results indicate that NO formation may play a role in QUIN-induced loss of enkephalinergic neurons, but not in the loss of NADPH diaphorase neurons.     

Distribution of GABA, glycine, and glutamate immunoreactivities in the vestibular nuclear complex of the frog

This study describes the localization of gamma-aminobutyric acid (GABA), glycine, and glutamate immunoreactive neurons, fibers, and terminal-like structures in the vestibular nuclear complex (VNC) of the frog by using a postembedding procedure with consecutive semithin sections at the light microscopic level. For purposes of this study, the VNC was divided into a medial and lateral region. Immunoreactive cells were observed in all parts of the VNC. GABA-positive neurons, generally small in size, were predominantly located in the medial part of the VNC. Glycine-positive cells, more heterogeneous in size than GABA-positive cells, were scattered throughout the VNC. A quantitative analysis of the spatial distribution of GABA glycine immunoreactive cells revealed a complementary relation between the density of GABA and glycine immunoreactive neurons along the rostrocaudal extent of the VNC. In about 10% of the immunolabeled neurons, GABA and glycine were colocalized. Almost all vestibular neurons were, to a variable degree, glutamate immunoreactive, and colocalization of glutamate with GABA and/or glycine was typical. GABA, glycine, or glutamate immunoreactive puncta were found in close contact to somata and main dendrites of vestibular neurons. A quantitative analysis revealed a predominance of glutamate-positive terminal-like structures compared to glycine or GABA containing profiles. A small proportion of terminal-like structures expressed colocalization of GABA and glycine or glycine and glutamate. The results are compared with data from mammals and discussed in relation to vestibuloocular and vestibulo-spinal projection neurons, and vestibular interneurons. GABA and glycine are the major inhibitory transmitters of these neurons in frogs as well as in mammals. The differential distribution of GABA and glycine might reflect a compartmentalization of neurons that is preserved to some extent from the early embryogenetic segmentation of the hindbrain.