Pupillary light reflex circuits in the macaque monkey: the preganglionic Edinger-Westphal nucleus.

The motor outflow for the pupillary light reflex originates in the preganglionic motoneuron subdivision of the Edinger-Westphal nucleus (EWpg), which also mediates lens accommodation. Despite their importance for vision, the morphology, ultrastructure and luminance-related inputs of these motoneurons have not been fully described in primates. In macaque monkeys, we labeled EWpg motoneurons from ciliary ganglion and orbital injections. Both approaches indicated preganglionic motoneurons occupy an EWpg organized as a unitary, ipsilateral cell column. When tracers were placed in the pretectal complex, labeled terminals targeted the ipsilateral EWpg and reached contralateral EWpg by crossing both above and below the cerebral aqueduct. They also terminated in the lateral visceral column, a ventrolateral periaqueductal gray region containing neurons projecting to the contralateral pretectum. Combining olivary pretectal and ciliary ganglion injections to determine whether a direct pupillary light reflex projection is present revealed a labeled motoneuron subpopulation that displayed close associations with labeled pretectal terminal boutons. Ultrastructurally, this subpopulation received synaptic contacts from labeled pretectal terminals that contained numerous clear spherical vesicles, suggesting excitation, and scattered dense-core vesicles, suggesting peptidergic co-transmitters. A variety of axon terminal classes, some of which may serve the near response, synapsed on preganglionic motoneurons. Quantitative analysis indicated that pupillary motoneurons receive more inhibitory inputs than lens motoneurons. To summarize, the pupillary light reflex circuit utilizes a monosynaptic, excitatory, bilateral pretectal projection to a distinct subpopulation of EWpg motoneurons. Furthermore, the interconnections between the lateral visceral column and olivary pretectal nucleus may provide pretectal cells with bilateral retinal fields.

The orbitofrontal cortex projects to the parvafox nucleus of the ventrolateral hypothalamus and to its targets in the ventromedial periaqueductal grey matter.

Although connections between the orbitofrontal cortex (OFC)-the seat of high cognitive functions-the lateral hypothalamus and the periaqueductal grey (PAG) have been recognized in the past, the precise targets of the descending fibres have not been identified. In the present study, viral tracer-transport experiments revealed neurons of the lateral (LO) and the ventrolateral (VLO) OFC (homologous to part of Area 13 in primates) to project to a circumscribed region in the ventrolateral hypothalamus, namely, the horizontally oriented, cylindrical parvalbumin- and Foxb1-expressing (parvafox) nucleus. The fine collaterals stem from coarse axons in the internal capsule and form excitatory synapses specifically with neurons of the parvafox nucleus, avoiding the rest of the hypothalamus. In its further caudal course, this contingent of LO/VLO-axons projects collaterals to the Su3- and the PV2 nuclei, which lie ventral to the aqueduct in the (PAG), where the terminals fields overlap those deriving from the parvafox nucleus itself. The targeting of the parvafox nucleus by the LO/VLO-projections, and the overlapping of their terminal fields within the PAG, suggest that the two cerebral sites interact closely. An involvement of this LO/VLO-driven circuit in the somatic manifestation of behavioural events is conceivable.

Dendritic morphology of caudal periaqueductal gray projecting retinal ganglion cells in Mongolian gerbil (Meriones unguiculatus).

In this study we investigated the morphological features of the caudal periaqueductal gray (cPAG)-projecting retinal ganglion cells (RGCs) in Mongolian gerbils using retrograde labeling, in vitro intracellular injection, confocal microscopy and three-dimensional reconstruction approaches. cPAG-projecting RGCs exhibit small somata (10-17 µm) and irregular dendritic fields (201-298 µm). Sizes of somata and dendritic fields do not show obvious variation at different distance from the optic disk (eccentricity). Dendrites are moderately branched. Morphological analysis (n = 23) reveals that cPAG-projecting RGCs ramified in sublamina a and b in the inner plexiform layer. These cells exhibit different stratification patterns based on the thickness of dendritic bands in sublaminas a and b: majority of analyzed cells (16 out of 23) have two bands of arborizations share similar thickness. The rest of analyzed cells (7 out of 23) exhibit thinner band in sublamina a than in sublamina b. Together, the present study suggests that cPAG of Mongolian gerbil could receive direct retinal inputs from two types of bistratified RGCs. Furthermore, a small subset of melanopsin-expressing RGCs (total 41 in 6 animals) is shown to innervate the rostral PAG (rPAG). Functional characteristics of these non-visual center projecting RGCs remain to be determined.

Columnar distribution of catecholaminergic neurons in the ventrolateral periaqueductal gray and their relationship to efferent pathways.

The periaqueductal gray (PAG) is a critical brain region involved in opioid analgesia and provides efferents to descending pathways that modulate nociception. In addition, the PAG contains ascending pathways to regions involved in the regulation of reward, including the substantia nigra (SN) and the ventral tegmental area (VTA). SN and VTA contain dopaminergic neurons that are critical for the maintenance of positive reinforcement. Interestingly, the PAG is also reported to contain a population of dopaminergic neurons. In this study, the distribution of catecholaminergic neurons within the ventrolateral (vl) PAG was examined using immunocytochemical methods. In addition, the catecholaminergic PAG neurons were examined to determine whether these neurons are integrated into ascending (VTA, SN) and descending rostral ventral medulla (RVM) efferent pathways from this region. The immunocytochemical analysis determined that catecholaminergic neurons in the PAG are both dopaminergic and noradrenergic and these neurons have a distinct rostrocaudal distribution within the ventrolateral column of PAG. Dopaminergic neurons were concentrated rostrally and were significantly smaller than noradrenergic neurons. Combined immunocytochemistry and tract tracing methods revealed that catecholaminergic neurons are distinct from, but closely associated with, both ascending and descending efferent projection neurons. Finally, by electron microscopy, catecholaminergic neurons showed close dendritic appositions with other neurons in PAG, suggesting a possible nonsynaptic mechanism for regulation of PAG output by these neurons. In conclusion, our data indicate that there are two populations of catecholaminergic neurons in the vlPAG that form dendritic associations with both ascending and descending efferents suggesting a possible nonsynaptic modulation of vlPAG neurons.

Spumiform basement membrane aberrations in the microvasculature of the midbrain periaqueductal gray region in hamster: rostro-caudal pathogenesis?

Spumiform basement membrane degeneration (sbmd) is a specific kind of aberration present in the capillaries of the midbrain periaqueductal gray (PAG) region of the senescent hamster. These capillaries, separated by the ependymal cell layer, are bordering the Sylvian cerebral aqueduct. The aqueduct, connecting the 3rd and 4th ventricle, may be crucial for local homeostatic as well as general autonomic functions of the PAG. Local pressure effects of the flowing and pulsating cerebrospinal fluid on the PAG-vasculature are probably different for the rostral 'entrance' and the caudal 'exit' of the aqueduct. In view of the different functions of the various divisions of the PAG, the frequency and extent of the aberrations in the rostral, intermediate and caudal dl/vlPAG-microvasculature could shed some light on the causal factors involved in the regional distribution of the particular microvascular aberrations found in the PAG during aging. In the present study we investigated the ultrastructure of capillaries in dorsal and ventral subdivisions of anterior and posterior regions of the PAG of young and old female Syrian hamsters. Sbmds were classified into four stages of spumiform severity and for each stage the frequency was determined in the rostral PAG, at two levels in the intermediate PAG and in a dorsal and a ventral part of the caudal PAG. Results of our quantitative studies showed that in aged hamster PAG various stages of sbmd were present in 91.6 ± 0.6% of all capillaries. No clear evidence was found for regional differentiation between rostral, intermediate and caudal parts of the PAG. Next to sbmd, capillary split basement membrane (sbm) and vacuolization were common features at all five PAG locations. 84.3 ± 2.3% of all screened PAG capillaries displayed sbm. In agreement with our previous findings, several other types of microvascular aberrations were observed in addition to general aspects of aging and some ependymal structural peculiarities. We conclude that the presence of various forms of sbmds in the PAG of senescent hamsters is a phenomenon that appears to be specific to the PAG region, but causal factors for this type of capillary degeneration remain unclear. Sbmds in the PAG may have serious consequences not only for blood-brain barrier functioning, but also for vascular perfusion and blood supply with eventually serious consequences for adequate regulation of the autonomic and motor control functions of the PAG region.

Ultrastructural analysis of rat ventrolateral periaqueductal gray projections to the A5 cell group.

Stimulation of neurons in the ventrolateral periaqueductal gray (PAG) produces antinociception as well as cardiovascular depressor responses that are mediated in part by pontine noradrenergic neurons. A previous report using light microscopy has described a pathway from neurons in the ventrolateral PAG to noradrenergic neurons in the A5 cell group that may mediate these effects. The present study used anterograde tracing and electron microscopic analysis to provide more definitive evidence that neurons in the ventrolateral PAG form synapses with noradrenergic and non-catecholaminergic A5 neurons in Sasco Sprague-Dawley rats. Deposits of anterograde tracer, biotinylated dextran amine, into the rat ventrolateral PAG labeled a significant number of axons in the region of the rostral subdivision of the A5 cell group, and a relatively lower number in the caudal A5 cell group. Electron microscopic analysis of anterogradely-labeled terminals in both rostral (n=127) and caudal (n=70) regions of the A5 cell group indicated that approximately 10% of these form synapses with noradrenergic dendrites. In rostral sections, about 31% of these were symmetric synapses, 19% were asymmetric synapses, and 50% were membrane appositions without clear synaptic specializations. In caudal sections, about 22% were symmetric synapses, and the remaining 78% were appositions. In both rostral and caudal subdivisions of the A5, nearly 40% of the anterogradely-labeled terminals formed synapses with non-catecholaminergic dendrites, and about 45% formed axoaxonic synapses. These results provide direct evidence for a monosynaptic pathway from neurons in the ventrolateral PAG to noradrenergic and non-catecholaminergic neurons in the A5 cell group. Further studies should evaluate if this established monosynaptic pathway may contribute to the cardiovascular depressor effects or the analgesia produced by the activation of neurons in the ventrolateral PAG.

Postnatal development of GABA-immunoreactive neurons and terminals in rat periaqueductal gray matter: a light and electron microscopic study.

The development of intrinsic gamma-aminobutyric acid (GABA)-ergic neurons was studied in the first month of postnatal life in the rat periaqueductal gray matter (PAG) by light and electron microscopy using an anti-GABA serum. At birth (postnatal day 0: P0) GABA-immunopositive (GABA(IP)) neurons were detected only on the outer edge of dorsolateral PAG (PAG-DL) and were rare in the other PAG subdivisions. Their distribution did not change from P0 to P5, while they increased progressively from P5 to P10 in PAG-DL and began to be detected in ventrolateral PAG (PAG-VL). At the end of the second postnatal week the immunostaining pattern was nearly adult-like, and between P20 and P30 the adult pattern of GABA immunoreactivity was established. Quantitative light microscopic examination indicated that in the first postnatal month the cross-sectional area of GABA(IP) neurons gradually increased from 67.63 and 78.69 microm(2) at P0 to 122.15 and 119.16 microm(2) at P30 in PAG-DL and PAG-VL, respectively. Electron microscopic observations disclosed GABA labeling from P0 in cell bodies, dendrites, growth cones, and axon terminals. GABA(IP) terminals were few in neonatal rats and became more numerous and morphologically mature around the second week. Synapse development and maturation were examined by quantitative ultrastructural analysis. Synaptic vesicle number and size of GABA(IP) axon terminals progressively grew in the first postnatal month. In conclusion, the number and size of GABA(IP) cells progressively increase in postnatal PAG, with two populations of intrinsic neurons expressing their GABAergic nature in two different periods.

Periaqueductal gray afferents synapse onto dopamine and GABA neurons in the rat ventral tegmental area.

The midbrain central gray (periaqueductal gray; PAG) mediates defensive behaviors and is implicated in the rewarding effects of opiate drugs. Projections from the PAG to the ventral tegmental area (VTA) suggest that this region might also regulate behaviors involving motivation and cognition. However, studies have not yet examined the morphological features of PAG axons in the VTA or whether they synapse onto dopamine (DA) or GABA neurons. In this study, we injected anterograde tracers into the rat PAG and used immunoperoxidase to visualize the projections to the VTA. Immunogold-silver labeling for tyrosine hydroxylase (TH) or GABA was then used to identify the phenotype of innervated cells. Electron microscopic examination of the VTA revealed axons labeled anterogradely from the PAG, including myelinated and unmyelinated fibers and axon varicosities, some of which formed identifiable synapses. Approximately 55% of these synaptic contacts were of the symmetric (presumably inhibitory) type; the rest were asymmetric (presumably excitatory). These findings are consistent with the presence of both GABA and glutamate projection neurons in the PAG. Some PAG axons contained dense-cored vesicles indicating the presence of neuropeptides in addition to classical neurotransmitters. PAG projections synapsed onto both DA and GABA cells with no obvious selectivity, providing the first anatomical evidence for these direct connections. The results suggest a diverse nature of PAG physiological actions on midbrain neurons. Moreover, as both the VTA and PAG are implicated in the reinforcing actions of opiates, our findings provide a potential substrate for some of the rewarding effects of these drugs.

Androgen and estrogen (alpha) receptor localization on periaqueductal gray neurons projecting to the rostral ventromedial medulla in the male and female rat.

The periaqueductal gray (PAG) is involved in many gonadal steroid-sensitive behaviors, including responsiveness to pain. The PAG projects to the rostral ventromedial medulla (RVM), comprising the primary circuit driving pain inhibition. Morphine administered systemically or directly into the PAG produces greater analgesia in male compared to female rats, while manipulation of gonadal hormones alters morphine potency in both sexes. It is unknown if these alterations are due to steroidal actions on PAG neurons projecting to the RVM. The expression of androgen (AR) and estrogen (ERalpha) receptors in the PAG of female rats and within this descending inhibitory pathway in both sexes is unknown. The present study used immunohistochemical techniques (1) to map the distribution of AR and ERalpha across the rostrocaudal axis of the PAG; and (2) to determine whether AR and/or ERalpha were colocalized on PAG neurons projecting to the RVM in male and female rats. AR and ERalpha immunoreactive neurons (AR-IR, ERalpha-IR) were densely distributed within the caudal PAG of male rats, with the majority localized in the lateral/ventrolateral PAG. Females had significantly fewer AR-IR neurons, while the quantity of ERalpha was comparable between the sexes. In both sexes, approximately 25-50% of AR-IR neurons and 20-50% of ERalpha-IR neurons were retrogradely labeled. This study provides direct evidence of the expression of steroid receptors in the PAG and the descending pathway driving pain inhibition in both male and female rats and may provide a mechanism whereby gonadal steroids modulate pain and morphine potency.

Mapping human whole-brain structural networks with diffusion MRI.

Understanding the large-scale structural network formed by neurons is a major challenge in system neuroscience. A detailed connectivity map covering the entire brain would therefore be of great value. Based on diffusion MRI, we propose an efficient methodology to generate large, comprehensive and individual white matter connectional datasets of the living or dead, human or animal brain. This non-invasive tool enables us to study the basic and potentially complex network properties of the entire brain. For two human subjects we find that their individual brain networks have an exponential node degree distribution and that their global organization is in the form of a small world.

TRPV1 receptor mediates glutamatergic synaptic input to dorsolateral periaqueductal gray (dl-PAG) neurons.

The purpose of this study was to determine the role of transient receptor potential vanilloid type 1 (TRPV1) receptor in modulating neuronal activity of the dorsolateral periaqueductal gray (dl-PAG) through excitatory and inhibitory synaptic inputs. First, whole cell voltage-clamp recording was performed to obtain the spontaneous miniature excitatory postsynaptic currents (mEPSCs) and inhibitory postsynaptic currents (mIPSCs) of the dl-PAG neurons. As 1 microM of capsaicin was applied into the perfusion chamber, the frequency of mEPSCs was increased from 3.21 +/- 0.49 to 5.64 +/- 0.64 Hz (P < 0.05, n = 12) without altering the amplitude and the decay time constant of mEPSCs. In contrast, capsaicin had no distinct effect on mIPSCs. A specific TRPV1 receptor antagonist, iodo-resiniferatoxin (i-RTX, 300 nM), decreased the frequency of mEPSCs from 3.51 +/- 0.29 to 2.01 +/- 0.2 Hz (P < 0.05, n = 8) but did not alter the amplitude and decay time. In addition, i-RTX applied into the chamber abolished the effect of capsaicin on mEPSC of the dl-PAG. In another experiment, spontaneous action potential of the dl-PAG neurons was recorded using whole cell current-clamp methods. Capsaicin significantly elevated the discharge rate of the dl-PAG neurons from 3.03 +/- 0.38 to 5.96 +/- 0.87 Hz (n = 8). The increased firing activity was abolished in the presence of glutamate N-methy-D-aspartate (NMDA) and non-NMDA antagonists, 2-amino-5-phosphonopentanoic acid, and 6-cyano-7-nitroquinoxaline-2,3-dione. The results from this study provide the first evidence indicating that activation of TRPV1 receptors increases the neuronal activity of the dl-PAG through selective potentiation of glutamatergic synaptic inputs.

Sumoylated RGS-Rz proteins act as scaffolds for Mu-opioid receptors and G-protein complexes in mouse brain.

The RGSZ1 and RGSZ2 proteins, members of the RGS-Rz subfamily of GTPase-activating proteins (GAP), are involved in Mu-opioid receptor desensitization. The expression of these proteins, as well as of their main target the Gz protein, is virtually restricted to the nervous tissue. In synaptosomal membranes, these Rz proteins undergo post-translational modifications such as glycosylation and phosphorylation, and they may covalently attach to small ubiquitin-like modifier (SUMO) proteins. While RGSZ1 exists in conjugated and non-conjugated forms, RGSZ2 is mostly conjugated to SUMO-1, SUMO-2 and SUMO-3 proteins. These sumoylated forms of the GAPs readily associated with Mu-opioid receptors but they associated only poorly with Delta receptors. Furthermore, G alpha i2 and G alpha z subunits co-precipitated with the sumoylated forms of RGSZ1/Z2 proteins, but to a lesser extent with the Ser phosphorylated SUMO-free form of RGSZ1. Upon Mu-opioid receptor activation, there is a strong increase in the association of G alpha proteins with RGSZ2 proteins that persists for intervals longer than 24 h. This effect probably accounts for their role in Mu-opioid receptor desensitization. Only a moderate increase was observed with RGSZ1, the non-sumoylated form of which probably acts as an efficient GAP for these G alpha subunits. Therefore, sumoylation regulates the biological activity of RGS-Rz proteins and it is likely that it serves to switch their behavior, from that of a GAP for activated G alpha subunits to that of a scaffold protein for specific signaling proteins.

A complete analysis of NMDA receptor subunits in periaqueductal grey and ventromedial medulla of morphine tolerant mice.

Behavioral studies indicate that the inhibition of glutamatergic system with antagonists of ionotropic glutamate NMDA receptors (NMDA-Rs) during long term morphine administration inhibits the development of morphine tolerance and dependence. In the present study we investigated whether chronic morphine treatment leading to the development of morphine antinociceptive tolerance as observed in tail-flick test in mice, may affect the expression of NMDA-R subunits. The expression of NMDA-R subunits was examined in brain areas mediating nociceptive signaling and responsible for antinociceptive activity of morphine. These included periaqueductal grey matter (PAG) and rostral ventromedial medulla (RVM). The expression of NR1 and all subunits of NR2 family (NR2A, NR2B, NR2C, NR2D) of NMDA receptor complex was examined using immunoblotting method. Although behavioral test indicated that morphine antinociceptive tolerance developed, changes in the expression of NMDA-R subunits were not observed, either in the PAG or in the RVM.

Differential roles of mGlu8 receptors in the regulation of glutamate and gamma-aminobutyric acid release at periaqueductal grey level.

We investigated the role of group III metabotropic glutamate (mGlu) receptors on glutamate and GABA releases at the periaqueductal grey (PAG) level by using in vivo microdialysis in rats. Intra-PAG perfusion of either L-(+)-2-amino-4-phosphonobutyric acid (L-AP4, 100-300 microM), (RS)-4-phosphonophenylglycine ((RS)-PPG, 100-300 microM) selective agonists of group III mGlu receptors, or (S)-3,4-dicarboxyphenylglycine ((S)-3,4-DCPG, 50-100 microM), a selective agonist of mGlu8 receptor, increased glutamate and decreased GABA extracellular concentrations. (RS)-alpha-methylserine-O-phosphate (MSOP, 0.5 mM), a selective group III receptor antagonist, perfused in combination with (S)-3,4-DCPG, L-AP4 or (RS)-PPG, antagonised the effects induced by these agonists on both extracellular glutamate and GABA values. alpha-Methyl-3-methyl-4-phosphonophenylglycine (UBP1112, 300 microM), a group III mGlu receptor antagonist, perfused in combination with (RS)-PPG or (S)-3,4-DCPG, antagonised the effects induced by these agonists. Intra-PAG perfusion with forskolin (100 microM), an activator of adenylate cyclase, increased dialysate glutamate and GABA levels. Moreover, intra-PAG perfusion with N-[2-(p-bromocinnamyl-amino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89) (100 microM), a protein kinase (PKA) inhibitor, abolished the effect of (S)-3,4-DCPG on both glutamate and GABA releases. H-89, per se, did not modify glutamate release but reduced extracellular GABA value at the higher dosage used (200 microM). These data suggest that group III mGlu receptors in the PAG modulate the releases of glutamate and GABA conversely. In particular, both the facilitation of glutamate and the inhibition of GABA releases require the participation of coupling to adenylate cyclase and the subsequent activation of the PKA pathway.

GABA-immunoreactive neurons and terminals in the cat periaqueductal gray matter: a light and electron microscopic study.

Immunocytochemical and electron microscopic methods were used to study the GABAergic innervation in adult cat periaqueductal gray matter (PAG). A mouse monoclonal antibody against gamma -aminobutyric acid (GABA) was used to visualize the inhibitory neuronal system of PAG. At light microscopy, GABA-immunopositive (GABA(IP)) neurons formed two longitudinally oriented columns in the dorsolateral and ventrolateral PAG that accounted for 36% of the neuronal population of both PAG columns; their perikaryal cross-sectional area was smaller than that of unlabeled (UNL) neurons found in the same PAG subdivisions. At electron microscopic level, patches of GABA immunoreactivity were readily detected in neuronal cell bodies, proximal and distal dendrites, axons and axon terminals. Approximately 35-36% of all terminals were GABA(IP); they established symmetric synapses with dendrites (84.72% of the sample in the dorsolateral PAG and 86.09% of the sample in the ventrolateral PAG) or with cell bodies (7-10% of the sample). Moreover, 49.15% of GABA(IP) axon terminals in the dorsolateral and 52.16% in the ventrolateral PAG established symmetric synapses with GABA(IP) dendrites. Immunopositive axon terminals and unlabeled terminals were also involved in the formation of a complex synaptic arrangment, i.e. clusters of synaptic terminals in close contact between them that were often observed in the PAG neuropil. Moreover, a fair number of axo-axonic synapses between GABA(IP) and/or UNL axon terminals were present in both PAG subdivisions. Several dendro-dendritic synapses between labeled and unlabeled dendrites were also observed in both PAG subdivisions. These results suggest that in the cat PAG there exist at least two classes of GABArgic neurons. The first class could exert a tonic control on PAG projecting neurons, the second could act on those GABAergic neurons that in turn keep PAG projecting neurons under tonic inhibition. The functional implications of this type of GABAergic synapse organization are discussed in relation to the dishinibitory processes that take place in the PAG.

[Projections of the dorsal raphe nucleus and midbrain central gray substance to the cat limbic system]. / Proektsii dorsal'nogo iadra shva i tsentral'nogo serogo veshchestva srednego mozga na limbicheskie struktury u koshki.

Morphological organization of connections of ventro-lateral (nociceptive) and dorso-lateral (analgetic) midbrain central gray (vl SGC and dl SGC), as well as of dorsal raphe nucleus (analgetic zone, Rd), with different limbic structures, responsible for the formation of various emotional states, was studied in 26 cats. The methods of electrical destruction of brain areas were used that were followed by the light and electron microscopic study of degenerating fibers and synapses. Heterogeneity of connections of above mentioned formations with different limbic structures was demonstrated. Connections Rd and dl SGC with upstream limbic structures were found to be very similar in their organization and expression. Connections of vl SGC with the same structures were significantly different. It is suggested that similar (antinociceptive) function of dl SGC and Rd has determined the likeness of their connections. This, in combination with the heterogeneity of SGC in conduction of the pain and analgesia, supports the identification of two brain systems: nociceptive, conducting pain sensitivity, and antinociceptive, inhibiting its conduction. The nociceptive system includes the following structures: vl SGC, posterior and lateral hypothalamic nuclei, preoptic area. In the antinociceptive system two subsystems could be distinguished: midbrain units of these subsystems are localized in different structures (Rd and dl SGC), while the upstream ones are found in the same hypothalamic nuclei--ventromedial, dorsomedial, paraventricular. As far as septum, amigdala, hippocampus and cingular cortex are concerned, it was found impossible to refer them to any of these systems--either nociceptive or antinociceptive--basing solely on the findings of morphological studies because of approximately similar representation of axons of neurons in vl SGC, dl SGC, Rd in these structures.

Translocation of presynaptic delta opioid receptors in the ventrolateral periaqueductal gray after swim stress.

Immunolabeling for the delta opioid receptor (DOR) is localized primarily to axon terminals in the ventrolateral periaqueductal gray (vlPAG). However, rather than on the plasma membrane, DOR immunoreactivity is usually located within the cytoplasmic compartment, often associated with dense-core vesicles. In this study, the hypothesis that a behavioral stimulus, a cold water swim stress (3 minutes at 4 degrees C; CWSS), could initiate the translocation of the DOR was tested. The subcellular distribution of DOR was examined using a preembedding immunogold-labeling method and ultrastructural analysis in control rats and in rats that had a CWSS. In both cases, dense-core vesicles associated with DOR labeling were often within 100 nm of the plasma membrane. When the dense-core vesicles were near the plasma membrane, sometimes electron-dense "tethers" appeared between the vesicle and the plasma membrane. However, in rats exposed to CWSS, there was a decrease in immunolabeling associated with dense-core vesicles that were near the plasma membrane and a significant increase in DOR immunoreactivity associated with the plasma membrane. In addition, there was a significant increase in the fraction of DOR immunoreactivity associated with large clear-core vesicles; possibly early endosomes. Moreover, after a CWSS, dense-core vesicles containing DOR immunoreactivity could be visualized fusing with the plasma membrane of synaptic boutons. These data suggest the involvement of DOR in the vlPAG in the behavioral response to CWSS. Furthermore, the results support the hypothesis that the cell surface distribution of presynaptic receptors can be regulated in an activity-dependent manner by virtue of transport via dense-core vesicles.

Group I metabotropic glutamate receptors modulate glutamate and gamma-aminobutyric acid release in the periaqueductal grey of rats.

In this study, we investigated the effects of group I metabotropic glutamate (mglu) receptor ligands on glutamate and gamma-aminobutyric acid (GABA) extracellular concentrations at the periaqueductal grey level by using in vivo microdialysis. An agonist of group I mglu receptors, (S)-3,5-dihydroxyphenylglycine [(S)-3,5-DHPG, 1 and 2 mM], as well as a selective agonist of mglu(5) receptors, (RS)-2-chloro-5-hydroxyphenylglycine (CHPG, 2 and 4 mM), both increased dialysate glutamate and GABA concentrations. 7-(Hydroxyimino)cyclopropa-[b]-chromen-1alpha-carboxylate ethyl ester (CPCCOEt, 1 mM), a selective mglu(1) receptor antagonist, and 2-methyl-6-(phenylethynyl)pyridine (MPEP, 0.5 mM), a selective mglu(5) receptor antagonist, perfused in combination with DHPG, antagonized the effect induced by DHPG on the extracellular glutamate and GABA concentrations. MPEP (0.5 mM), perfused in combination with CHPG, antagonized the increased glutamate and GABA extracellular levels induced by CHPG. MPEP (1 mM) decreased the extracellular concentrations of glutamate but did not modify the dialysate GABA concentrations. Moreover, as the intra-periaqueductal grey perfusion of (RS)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid [(RS)-CPP, 100 microM], a selective N-methyl-D-aspartate (NMDA) glutamate receptor antagonist, did not change the extracellular concentrations of glutamate, this suggests that the MPEP-induced decrease in glutamate is not a consequence of NMDA receptor blockade. These data show that group I mglu receptors in the periaqueductal grey may modulate the release of glutamate and GABA in awake, freely moving rats. In particular, mglu(5), but not mglu(1), receptors seem to be functionally active on glutamate terminals.

MR imaging assessment of myelination in the very preterm brain.

BACKGROUND AND PURPOSE: MR imaging was performed in very preterm infants by using an MR imager in the neonatal intensive care unit. The aims of this study were to assess the development of myelination in the preterm brain based on MR imaging findings and to compare the ability of T1-weighted conventional spin-echo, inversion recovery fast spin-echo, and T2-weighted fast spin-echo MR imaging to show myelination in these infants. METHODS: MR imaging was performed for 26 preterm infants with a median gestational age of 28 weeks who had normal neurodevelopmental outcomes at 2 years corrected age. RESULTS: Myelin was evident in the gracile and cuneate nuclei and fasciculi, vestibular nuclei, cerebellar vermis, inferior and superior cerebellar peduncles, dentate nucleus, medial longitudinal fasciculus, medial geniculate bodies, subthalamic nuclei, inferior olivary nuclei, ventrolateral nuclei of the thalamus, decussation of the superior cerebellar peduncles, medial lemnisci, lateral lemnisci, and inferior colliculi at < or = 28 weeks gestational age. From this gestational age, myelination was not visualized at any new site until 36 weeks gestational age, when myelin was visualized in the corona radiata, posterior limb of the internal capsule, corticospinal tracts of the precentral and postcentral gyri, and lateral geniculate bodies. T2-weighted fast spin-echo MR imaging showed myelin in gray matter nuclei at an earlier gestational age than did T1-weighted conventional spin-echo or inversion recovery fast spin-echo MR imaging. T1-weighted conventional spin-echo MR imaging showed myelin earlier in some white matter tracts in the preterm brain. CONCLUSION: Myelination was evident in numerous gray and white matter structures in the very preterm brain. A knowledge of myelination milestones will allow delays to be detected at an early stage.

Cellular basis for the effects of substance P in the periaqueductal gray and dorsal raphe nucleus.

Substance P (SP) is known to act at supraspinal sites to influence pain sensitivity as well as to promote anxiety. The effects of SP could be mediated in part by actions in the periaqueductal gray (PAG) and the dorsal raphe nucleus (DRN), adjoining mesencephalic cell groups that are strategically positioned to influence both nociception and mood. Previous studies have indicated that SP regulates both enkephalin and serotonin neurotransmission in these brain regions. To determine the mechanism underlying the effects of SP in the PAG and DRN, the distribution of the principal receptor for SP, the neurokinin 1 (NK1) receptor, was examined with respect to other neurotransmitter markers. PAG neurons that had NK1 receptor immunolabeling were interdigitated with and received contacts from enkephalin-containing neurons. However, only a few (16/144; 11%) neurons with NK1 receptor also contained enkephalin immunoreactivity after colchicine treatment. In the DRN, dendrites containing NK1 receptor were selectively distributed in the dorsomedial subdivision. The majority (132/137; 96%) of these dendrites did not contain immunoreactivity for the serotonin-synthesizing enzyme tryptophan hydroxylase. In contrast, neuronal profiles with NK1 receptor in both the PAG and the DRN often contained immunolabeling for glutamate. Light and electron microscopic examination revealed that 48-65% of cell bodies and dendrites with NK1 receptor were dually immunolabeled for glutamate. These data suggest that SP directly acts primarily on glutamatergic neurons in the PAG and DRN. To a lesser extent, enkephalin-containing neurons may be targeted. Through these actions, it may subsequently influence activity of larger populations of neurons containing enkephalin as well as serotonin. This circuitry could contribute to, as well as coordinate, effects of SP on pain perception and mood.