Lysosomal response in relation to α-synuclein pathology differs between Parkinson's disease and multiple system atrophy.

Intracellular deposition of pathologically altered α-synuclein mostly in neurons characterises Parkinson's disease (PD), while its accumulation predominantly in oligodendrocytes is a feature of multiple system atrophy (MSA). Recently a prion-like spreading of pathologic α-synuclein has been suggested to play a role in the pathogenesis of PD and MSA. This implicates a role of protein processing systems, including lysosomes, supported also by genetic studies in PD. However, particularly for MSA, the mechanism of cell-to-cell propagation of α-synuclein is yet not fully understood. To evaluate the significance of lysosomal response, we systematically compared differently affected neuronal populations in PD, MSA, and non-diseased brains using morphometric immunohistochemistry (cathepsin D), double immunolabelling (cathepsin D/α-synuclein) laser confocal microscopy, and immunogold electron microscopy for the disease associated α-synuclein. We found that i) irrespective of the presence of neuronal inclusions, the volume density of cathepsin D immunoreactivity significantly increases in affected neurons of the pontine base in MSA brains; ii) volume density of cathepsin D immunoreactivity increases in nigral neurons in PD without inclusions and with non-ubiquitinated pre-aggregates of α-synuclein, but not in neurons with Lewy bodies; iii) cathepsin D immunoreactivity frequently colocalises with α-synuclein pre-aggregates in nigral neurons in PD; iv) ultrastructural observations confirm disease-associated α-synuclein in neuronal and astrocytic lysosomes in PD; v) lysosome-associated α-synuclein is observed in astroglia and rarely in oligodendroglia and in neurons in MSA. Our observations support a crucial role for the neuronal endosomal-lysosomal system in the processing of α-synuclein in PD. We suggest a distinct contribution of lysosomes to the pathogenesis of MSA, including the possibility of oligodendroglial and eventually neuronal uptake of exogenous α-synuclein in MSA.

[Structural basis for the inhibitory function of the parietal cortex efferent systems].

Relative quantitative distribution of all the associative and descending efferent fibers and the ultrastructural organization of the terminals of the parietal cortex areas 5 and 7 in the caudate (NC) and red nucleus (NR) in the cat were analyzed after a local, pointed destruction of the cortex of these areas. The maximal numbers of the associative fibers were found to project to the fundus areas of the motor cortex and to the area of Clare-Bishop; moderate projections were detected to the areas 31, 19 and single degenerating fibers were registered in the areas 1,2, 3a, 3b, 30, and 23. The descending efferents were maximally projecting to NC, NR, reticular nuclei of the thalamus, midbrain, and pons, in all of which, according to the immunocytochemical studies, GABA-ergic terminals are prevalent. On the basis on the electron microscopical studies, it was suggested that the influence of the parietal cortex is mediated by the axo-spinal synapses of the medium shortaxonal spiny cells of the dorsolateral part of NC caput and by the axo-dendritic synapses of Golgi II cells of the parvocellular part of NR. On the basis of the maximal involvement of the fundus areas of the motor cortex, as well as of the inhibitory subcortical (NC) and stem nuclei (NR, reticular nuclei of the thalamus, midbrain, and nuclei pontis), it is suggested that these structures serve as the morphological substrates for the realization of the inhibitory, integrative function of the parietal cortex.

Interactions between orexin-immunoreactive fibers and adrenaline or noradrenaline-expressing neurons of the lower brainstem in rats and mice.

Orexins are expressed in neurons of the dorsolateral hypothalamus and their axons widely distribute throughout the central nervous system. The noradrenergic cell groups of the lower brainstem belong to the targets of these orexin projections. Double immunostainings for orexin and phenylethanolamine N-methyltransferase (PNMT), as well as orexin and tyrosine hydroxylase (TH) were applied to demonstrate the orexinergic innervation of catecholamine cell groups in the lower brainstem of the mouse and the rat. In various densities, networks of orexin-positive fibers and terminals were present on neurons of each adrenaline (C1, C2, C3) and noradrenaline (locus coeruleus, A1, A2, A4, A5 and A7) cell groups. The most dense networks of orexin fibers and terminals were detected in the locus coeruleus, the subcoeruleus area, and in the nucleus of the solitary tract. By using confocal microscope to analyze triple immunostainings we could detect that about two-third of the orexin-PNMT or orexin-TH immunopositive close contacts contained synaptophysin (a presynapse-specific protein) in the C1, C2 and C3 adrenaline, or in the A1, A2 noradrenaline cell groups, respectively. Orexin-immunopositive axons in the C1, C2, as well as A1, A2 and A6 cell groups have been examined by an electron microscope. Relatively few asymmetrical (excitatory) synaptic contacts could be demonstrated between PNMT- or TH-positive dendrites and orexin terminals, although the vast majority of orexin-positive axons was located in juxtaposition to PNMT- or TH-positive neurons.

The micro-architecture of mitochondria at active zones: electron tomography reveals novel anchoring scaffolds and cristae structured for high-rate metabolism.

Mitochondria are integral elements of many nerve terminals. They must be appropriately positioned to regulate microdomains of Ca(2+) concentration and metabolic demand, but structures that anchor them in place have not been described. By applying the high resolution of electron tomography (ET) to the study of a central terminal, the calyx of Held, we revealed an elaborate cytoskeletal superstructure that connected a subset of mitochondria to the presynaptic membrane near active zones. This cytoskeletal network extended laterally and was well integrated into the nerve terminal cytoskeleton, which included filamentous linkages among synaptic vesicles. ET revealed novel features of inner membrane for these mitochondria. Crista structure was polarized in that crista junctions, circular openings of the inner membrane under the outer membrane, were aligned with the cytoskeletal superstructure and occurred at higher density in mitochondrial membrane facing the presynaptic membrane. These characteristics represent the first instance where a subcomponent of an organelle is shown to have a specific orientation relative to the polarized structure of a cell. The ratio of cristae to outer membrane surface area is large in these mitochondria relative to other tissues, indicating a high metabolic capacity. These observations suggest general principles for cytoskeletal anchoring of mitochondria in all tissues, reveal potential routes for nonsynaptic communication between presynaptic and postsynaptic partners using this novel cytoskeletal framework, and indicate that crista structure can be specialized for particular functions within cellular microdomains.

Ultrastructural characterization of relationship between serotonergic and GABAergic structures in the ventral part of the oral pontine reticular nucleus.

The ventral part of the oral pontine reticular nucleus (vRPO) is involved in the generation and maintenance of rapid eye movement (REM) sleep. Both GABAergic and serotonergic neurotransmission have been implicated in the control of the sleep-wakefulness cycle. Nevertheless, the synaptic organization of serotonergic terminals in the vRPO has not yet been characterized. We performed an electron microscope study of serotonin-immunoreactive (5-HT-IR) terminals using immunoperoxidase or immunogold-silver methods. In a second set of experiments, combining GABA immunoperoxidase and 5-HT immunogold-silver techniques, we examined inputs from GABA-immunoreactive (GABA-IR) terminals to serotonergic neurons. 5-HT-IR terminals were located primarily on dendrites and occasionally on somata of unlabeled and 5-HT-IR neurons. The majority of the synapses formed by 5-HT-IR terminals were of the symmetrical type, making contacts primarily with unlabeled dendritic profiles. Moreover, 5-HT-IR terminals contacted unlabeled axon terminals that formed asymmetric synapses on dendrites. Double immunolabeling experiments showed 5-HT-IR and GABA-IR afferents, in apposition to each other, making synapses with the same dendrites. Finally, GABA-IR terminals innervated 5-HT-IR and GABA-IR dendrites. Our findings indicate that serotonin would modulate the neuronal activity through inhibitory or excitatory influences, although the action of serotonin on the vRPO would predominantly be inhibitory. Moreover, the present results suggest that the serotonin modulation of vRPO neurons might involve indirect connections. In addition, GABA might contribute to the induction and maintenance of REM sleep by inhibiting serotonergic and GABAergic neurons in the vRPO.

Dynorphin-A immunoreactive terminals on the neuronal somata of rat mesencephalic trigeminal nucleus.

Dynorphin-A-like immunoreactivity was investigated in the rat mesencephalic trigeminal nucleus (Mes 5) at the light and electron microscopic levels. Dynorphin-A immunoreactive fibers and puncta, likely representing nerve terminals, were observed throughout rostrocaudal extension of the Mes 5 at the light microscopic level. Within the rostrocaudal extension, more abundant fibers and puncta were localized in the midbrain-pontine junction and pontine areas than in the midbrain area. At the electron microscopic level, dynorphin-A immunoreactive synapses were observed on the somata of Mes 5. Dynorphin-A-like immunoreactivity tended to be restricted to dense-cored vesicles in the synapses. These results suggest that dynorphin-A-containing fiber systems affect mastication through the Mes 5.

GABAergic structures in the ventral part of the oral pontine reticular nucleus: An ultrastructural immunogold analysis.

GABA mediates inhibitory effects in neurons of the ventral part of the oral pontine reticular nucleus (vRPO). Evidence increasingly suggests that GABA plays an important role in the modulation of rapid eye movement (REM) sleep generation in the cat vRPO. Here, we investigate the anatomical substrate of this modulation using GABA immunocytochemistry. Immunoperoxidase labeling revealed a few small GABA-immunoreactive cell bodies scattered throughout the vRPO. The numerical densities of all vRPO synapses and the GABA-immunoreactive synapses were estimated, at the electron microscopical level, by using a combination of the physical disector and the post-embedding immunogold techniques. We estimated that 30% of all vRPO synaptic terminals were immunoreactive to GABA. Our findings support the hypothesis that vRPO neuron activity is significantly controlled by inhibitory GABAergic terminals that directly target somata and the different parts of the dendritic tree, including distal regions. GABAergic input could inhibit vRPO REM sleep-inducing neurons during other states of the sleep-wakefulness cycle such as wakefulness or non-REM sleep.

Trak1 mutation disrupts GABA(A) receptor homeostasis in hypertonic mice.

Hypertonia, which results from motor pathway defects in the central nervous system (CNS), is observed in numerous neurological conditions, including cerebral palsy, stroke, spinal cord injury, stiff-person syndrome, spastic paraplegia, dystonia and Parkinson disease. Mice with mutation in the hypertonic (hyrt) gene exhibit severe hypertonia as their primary symptom. Here we show that hyrt mutant mice have much lower levels of gamma-aminobutyric acid type A (GABA(A)) receptors in their CNS, particularly the lower motor neurons, than do wild-type mice, indicating that the hypertonicity of the mutants is likely to be caused by deficits in GABA-mediated motor neuron inhibition. We cloned the responsible gene, trafficking protein, kinesin binding 1 (Trak1), and showed that its protein product interacts with GABA(A) receptors. Our data implicate Trak1 as a crucial regulator of GABA(A) receptor homeostasis and underscore the importance of hyrt mice as a model for studying the molecular etiology of hypertonia associated with human neurological diseases.

Branching patterns of parabrachial neurons projecting to the central extended amgydala: single axonal reconstructions.

Electrophysiological evidence suggests that the spinoparabrachioamygdaloid pathway carries nociceptive information that may be important for the elaboration of physiological and emotional responses to noxious events. The pontine parabrachial nucleus (pPB) sends a massive projection to the central nucleus of the amygdala (CeA) and lateral bed nucleus of the stria terminalis (BSTL), both regions belonging to a broader macrostructure, the central extended amygdala (EAc). The aim of this study was to examine whether different EAc components are targeted by a same pPB neuron, by reconstructing single axonal branching patterns after anterograde labelling. Small deposits of biotinylated dextran amine in the region of the external lateral pPB result in dense and specific labelling in the whole EAc. Reconstructed axons innervate either the lateral or the capsular part of the CeA with perisomatic or bushy terminals, respectively. A subset of axons enters the stria terminalis rostrally to follow its trajectory caudally toward the CeA. Individual axons targeting the CeA usually send collaterals to other EAc components, especially those projecting to the lateral CeA, which often coinnervate the BSTL. By contrast, only few branches were found outside the EAc. These results suggest that the noxious information travelling from the pPB to the CeA may also be transmitted to other EAc components. This pPB-EAc pathway, which appears distinct from the parabrachiohypothalamic and parabrachiothalamic projections, would be the anatomical basis through which the EAc elaborates the autonomic, endocrine, and emotional components of pain.

Neurokininergic mechanism within the lateral crescent nucleus of the parabrachial complex participates in the heart-rate response to nociception.

We wanted to ascertain whether the lateral parabrachial nucleus was involved in mediating the heart-rate response evoked during stimulation of somatic nociceptors. Reversible inactivation of the lateral parabrachial nucleus, using a GABA(A) agonist, reduced the reflex tachycardia evoked during noxious (mechanical) stimulation of the forelimb by approximately 50%. The same effect was observed after blockade of neurokinin 1 receptors within the lateral parabrachial nucleus, indicating a possible involvement for substance P as a neurotransmitter. Immunocytochemistry revealed a strong expression of substance P-immunoreactive fibers and boutons in all lateral subnuclei, but they were particularly dense in the lateral crescent subnucleus. Histological verification showed that the most effective injection sites for attenuating the noxious-evoked tachycardia were all placed in or near to the lateral crescent nucleus of the lateral parabrachial complex. Many single units recorded from this region were activated by high-intensity brachial nerve stimulation. The brachial nerve evoked firing responses of some of these neurons was reversibly reduced after local delivery of a neurokinin 1 receptor antagonist. However, only a minority of these neurons followed a paired-pulse stimulation protocol applied to the spinal cord, suggesting a predominance of indirect projections from the spinal cord to the parabrachial nucleus. We conclude that the cardiac component of the response to somatic nociception involves indirect spinal pathways that most likely excite neurons located in the lateral crescent nucleus of the parabrachial complex via activation of neurokinin 1 receptors.

Laterodorsal tegmental projections to identified cell populations in the rat ventral tegmental area.

Projections from the laterodorsal tegmentum (LDT) to the ventral tegmental area (VTA) contribute to the activity of dopamine (DA) and GABA cells and, hence, to the affective and cognitive functions of this region. LDT afferents arise from neurochemically diverse cell types and mediate multiple functional influences. However, the VTA cell populations that receive LDT afferents are unknown and were investigated here by anterograde and retrograde tract-tracing in combination with immunocytochemistry to distinguish DA and GABA cells. Approximately 50% of the LDT to VTA pathway formed asymmetric, presumably excitatory synapses that innervated DA and GABA cells in rough proportion to their representation within the VTA. This portion of the LDT innervation appeared to selectively target DA but not GABA mesoaccumbens neurons and provide a relatively nonselective input to both DA and GABA mesoprefrontal cells. The remaining LDT axons formed symmetric, presumably inhibitory synapses with a different pattern of cellular targets that included a preferential input to GABA neurons of both mesoaccumbens and mesoprefrontal populations and an apparently selective innervation of mesoprefrontal and not mesoaccumbens DA neurons. These data suggest that the LDT mediates a convergent excitatory and inhibitory influence on both mesoprefrontal DA and GABA cells but a divergent impact on mesoaccumbens neurons that is likely to excite DA cells and inhibit GABA neurons. Combined with our previous description of prefrontal cortical afferents, our data also indicate that mesoaccumbens DA neurons receive putative excitatory drive from the LDT, whereas mesoprefrontal DA cells receive convergent excitation from both cortical and brainstem sources.

Rat trigeminal lamina I neurons that project to thalamic or parabrachial nuclei contain the mu-opioid receptor.

Ligands of the mu-opioid receptor are known to inhibit nociceptive transmission in the dorsal horn, yet the cellular site(s) of action for this inhibition remain to be fully elucidated. Neurons located in lamina I of the dorsal horn are involved in distinct aspects of nociceptive transmission. Neurons projecting to the thalamus are thought to be involved in sensory-discriminative aspects of pain perception, while neurons projecting to the parabrachial nucleus are thought to be important for emotional and/or autonomic responses to noxious stimuli. The present study examined these two populations of lamina I projection neurons in the trigeminal dorsal horn to determine if the mu-opioid receptor protein (MOR1) is differentially located in these populations of neurons. Lamina I projection neurons were identified using the retrograde tracer FluoroGold (FGold). FGold was injected into either the contralateral thalamus (ventral posterolateral (VPM)/ventral posterolateral (VPL) thalamic region) or into the ipsilateral parabrachial nuclei. The distribution of MOR1 in these neurons was determined using immunocytochemistry. The distribution of MOR1-ir within these two populations of lamina I projection neurons was examined by both confocal and electron microscopy. We found that both populations of projection neurons contained MOR1. Immunogold analyses revealed the presence of MOR1-ir at membrane sites and within the cytoplasm of these neurons. Cytoplasmic receptor labeling may represent sites of synthesis, recycling or reserve populations of receptors. MOR1 was primarily found in the somata and proximal dendrites of projection neurons. In addition, these neurons rarely received synaptic input from MOR1-containing axon terminals. These results indicate that lamina I neurons in trigeminal dorsal horn that project to the thalamic and parabrachial nuclei contain MOR1 and are likely sites of action for MOR ligands that modulate sensory and/or autonomic aspects of pain transmission in the trigeminal dorsal horn.

Parabrachial inputs to Fos-immunoreactive neurons in the lateral central nucleus of amygdala activated by hypotension: a light and electron microscopic study in the rat.

Morphological features and functional implications of projections of the parabrachial nucleus to the central nucleus of the amygdala were investigated in the rat. The anatomical study was based on injections of the tracers horseradish peroxidase and biotinylated dextran amine. An extremely dense concentration of labeled fibers was found in the lateral and lateral capsular subdivisions of the central nucleus of the amygdala, originating mainly from the external lateral and ventral lateral subnuclei of the parabrachial nucleus. The parabrachial fibers exhibited the morphological characteristic of forming dense pericellular terminal arborizations. The functional implications of this pathway in cardiovascular functions were verified using Fos protein induction in response to hypotension induced by continuous intravenous administration of hydralazine-hydrochloride. In this paradigm, Fos immunoreactivity was found to be confined to the lateral and lateral capsular subdivisions of the central nucleus of the amygdala. Double immunostaining methods were used to visualize, at the electron microscopic level, terminals labeled by biotinylated dextran amine and Fos cell labeling. With this approach, we were able to confirm that Fos-immunoreactive neurons in the central nucleus of the amygdala receive axosomatic terminals from the parabrachial nucleus. The present findings point out that parabrachial inputs to the central nucleus of the amygdala play a relevant role in regulating cardiovascular function.

Mapping and quantitative analysis of gephyrin cytoplasmic trafficking pathways in motoneurons, using an optimized Transmission Electron Microscopy Color Imaging (TEMCI) procedure.

In the present study, an optimized Transmission Electron Microscopy Color Imaging (TEMCI) procedure was used to map and quantify the pathways involved in the trafficking and subcellular targeting of gephyrin in identified abducens motoneurons. Gephyrin is a scaffolding protein, which plays a crucial role in the clustering of the GABA(A) and glycine receptors to the cytoskeleton. TEMCI associated several accurate tools: (i) nanogold immunodetection of gephyrin in motoneurons identified on the basis of their immunoreactivity to Choline Acetyl Transferase, (ii) low magnification color scale coding of gephyrin densities on series of ultrathin sections of motoneurons, which gave a map of the cytoplasmic distribution of the protein, (iii) statistical analysis of the subcellular distribution of the immunolabeling. The color map of gephyrin densities in the cell bodies reflected the distribution of inhibitory synapses over the membrane. The TEMCI analysis of motoneurons with various patterns of synaptic covering made it possible to visualize for the first time the cytoplasmic transport pathway of gephyrin towards its target at synaptic contact. A high magnification quantitative analysis, including the study of 109 inhibitory synapses, showed that most gephyrin-associated immunogold particles (67%) were located in the subsynaptic regions facing the active zones, and the second most densely occupied regions were the perisynaptic regions (19.5% of immunogold particles). A consistent proportion of the gephyrin (11.5%), significantly higher than densities present in the rest of the cytoplasm (2%), was detected in the extrasynaptic submembrane region.

Acute moderate hyponatraemia and its rapid correction: effects on striatal and pontine ultrastructure in an animal model of the TURP syndrome.

BACKGROUND AND OBJECTIVE: To investigate the effects of moderate hyponatraemia, induced by intravenous application of an electrolyte-free irrigation fluid, as a model of the human transurethral prostate resection syndrome and of its rapid correction by hypertonic saline infusion in rats. METHODS: Experimental animals received irrigation fluid (Purisole SM) 20 mL kg(-1) body weight, intravenously. In one group, hyponatraemia was subsequently rapidly corrected by infusion of hypertonic saline (NaCl 5.85%), while rats of group two were 'sham-corrected' by infusion of a balanced salt crystalloid solution. Plasma sodium concentrations were analysed during and at the end of the experiments. After 10 days, experimental and untreated control animals were killed humanely, fixed by perfusion and the brains were prepared for electron microscopic investigation of myelin sheets and glial cell numbers in the striatum and pons. RESULTS: The myelin appearance was unaltered in experimental groups compared to controls, but glial cell numbers were distinctly altered in the pons but not in the striatum. In the pons, oligodendrocytes were significantly reduced in number upon rapid correction of hyponatraemia, while astrocyte numbers were increased in rats with uncorrected hyponatraemia. CONCLUSIONS: Our electron microscopic data demonstrate that the effects of hyponatraemia and of its rapid correction are multifarious in animals. This may also apply for human patients during transurethral prostate resection.

Prefrontal cortical projections to the rat dorsal raphe nucleus: ultrastructural features and associations with serotonin and gamma-aminobutyric acid neurons.

Studies of human brain indicate that both the ventromedial prefrontal cortex (PFC) and the dorsal raphe nucleus (DRN) may be dysfunctional in major depressive illness, making it important to understand the functional interactions between these brain regions. Anatomical studies have shown that the PFC projects to the DRN, although the synaptic targets of this excitatory pathway have not yet been identified. Electrophysiological investigations in the rat DRN report that most serotonin neurons are inhibited by electrical stimulation of the PFC, suggesting that this pathway is more likely to synapse onto neighboring gamma-aminobutyric acid (GABA) neurons than onto serotonin cells. We tested this hypothesis by electron microscopic examination of DRN sections dually labeled for biotin dextran amine anterogradely transported from the PFC and immunogold-silver labeling for tryptophan hydroxylase (TrH) or for GABA. In the DRN, the majority of PFC axons either synapsed onto unlabeled dendrites or failed to form detectable synapses in single sections. Other PFC axons synapsed onto either TrH- or GABA-immunolabeled processes. Considerably more tissue sampling was necessary to detect PFC synapses onto TrH- than onto GABA-labeled dendrites, suggesting that the latter connections are more common. In other cases, PFC terminals and TrH- or GABA-immunoreactive dendrites either were closely apposed, without forming detectable synapses, or were separated by glial processes. These results provide potential anatomical substrates whereby the PFC can both directly and indirectly regulate the activity of serotonin neurons in the DRN and possibly contribute to the pathophysiology of depression.

Cellular interactions between axon terminals containing endogenous opioid peptides or corticotropin-releasing factor in the rat locus coeruleus and surrounding dorsal pontine tegmentum.

Recent evidence suggests that certain stressors release both endogenous opioids and corticotropin-releasing factor (CRF) to modulate activity of the locus coeruleus (LC)-norepinephrine (NE) system. In ultrastructural studies, axon terminals containing methionine(5)-enkephalin (ENK) or CRF have been shown to target LC dendrites. These findings suggested the hypothesis that both neuropeptides may coexist in common axon terminals that are positioned to have an impact on the LC. This possibility was examined by using immunofluorescence and immunoelectron microscopic analysis of the rat LC and neighboring dorsal pontine tegmentum. Ultrastructural analysis indicated that CRF- and ENK-containing axon terminals were abundant in similar portions of the neuropil and that approximately 16% of the axon terminals containing ENK were also immunoreactive for CRF. Dually labeled terminals were more frequently encountered in the "core" of the LC vs. its extranuclear dendritic zone, which included the medial parabrachial nucleus (mPB). Triple labeling for ENK, CRF, and tyrosine hydroxylase (TH) showed convergence of opioid and CRF axon terminals with noradrenergic dendrites as well as evidence for inputs to TH-labeled dendrites from dually labeled opioid/CRF axon terminals. One potential source of ENK and CRF in the dorsal pons is the central nucleus of the amygdala (CNA). To determine the relative contribution of ENK and CRF terminals from the CNA, the CNA was electrolytically lesioned. Light-level densitometry revealed robust decreases in CRF immunoreactivity in the LC and mPB on the side ipsilateral to the lesion but little or no change in ENK immunoreactivity, confirming previous studies of the mPB. Degenerating terminals from the CNA in lesioned rats were found to be in direct contact with TH-labeled dendrites. Together, these data indicate that ENK and CRF may be colocalized to a subset of individual axon terminals in the LC "core." The finding that the CNA provides, to dendrites in the area examined, a robust CRF innervation, but little or no opioid innervation, suggests that ENK and CRF axon terminals impacting LC neurons originate from distinct sources and that terminals that colocalize ENK and CRF are not from the CNA.

A novel central pathway links arterial baroreceptors and pontine parasympathetic neurons in cerebrovascular control.

1. We tested the hypothesis that arterial baroreceptor reflexes modulate cerebrovascular tone through a pathway that connects the cardiovascular nucleus tractus solitarii with parasympathetic preganglionic neurons in the pons. 2. Anesthetized rats were used in all studies. Laser flowmetry was used to measure cerebral blood flow. We assessed cerebrovascular responses to increases in arterial blood pressure in animals with lesions of baroreceptor nerves, the nucleus tractus solitarii itself, the pontine preganglionic parasympathetic neurons, or the parasympathetic ganglionic nerves to the cerebral vessels. Similar assessments were made in animals after blockade of synthesis of nitric oxide, which is released by the parasympathetic nerves from the pterygopalatine ganglia. Finally the effects on cerebral blood flow of glutamate stimulation of pontine preganglionic parasympathetic neurons were evaluated. 3. We found that lesions at any one of the sites in the putative pathway or interruption of nitric oxide synthesis led to prolongation of autoregulation as mean arterial pressure was increased to levels as high as 200 mmHg. Conversely, stimulation of pontine parasympathetic preganglionic neurons led to cerebral vasodilatation. The second series of studies utilized classic anatomical tracing methods to determine at the light and electron microscopic level whether neurons in the cardiovascular nucleus tractus solitarii, the site of termination of baroreceptor afferents, projected to the pontine preganglionic neurons. Fibers were traced with anterograde tracer from the nucleus tractus solitarii to the pons and with retrograde tracer from the pons to the nucleus tractus solitarii. Using double labeling techniques we further studied synapses made between labeled projections from the nucleus tractus solitarii and preganglionic neurons that were themselves labeled with retrograde tracer placed into the pterygopalatine ganglion. 4. These anatomical studies showed that the nucleus tractus solitarii directly projects to pontine preganglionic neurons and makes asymmetric, seemingly excitatory, synapses with those neurons. These studies provide strong evidence that arterial baroreceptors may modulate cerebral blood flow through direct connections with pontine parasympathetic neurons. Further study is needed to clarify the role this pathway plays in integrative physiology.

Subcellular localization of the voltage-dependent potassium channel Kv3.1b in postnatal and adult rat medial nucleus of the trapezoid body.

A pre-embedding immunocytochemical method was used to study the subcellular distribution of the voltage-dependent potassium channel Kv3.1b in the medial nucleus of the trapezoid body (MNTB) in developing and adult rat. The main finding was the localization of the channel in specific membrane compartments of the calyces of Held and principal globular neurons. Thus, at postnatal day (P) 9 immunoparticles were densely localized in plasma membranes of globular cell bodies and their main dendrites. At P16, a strong Kv3.1b labeling was still observed in these globular cell compartments, but the most remarkable feature was the presence of immunoparticles in synaptic terminal membranes of the calyces of Held. However, the presynaptic and postsynaptic specializations of the calyx of Held-globular cell synapses were virtually devoid of immunoparticles. This same subcellular distribution of Kv3.1b was seen in adult, with membranes of calycine terminals more uniformly labeled. The developmental profile of Kv3.1b expression in MNTB coincides with the functional maturation of the calyx of Held-principal globular neuron synapse. The presence of the channel in this system is crucial for the high-frequency synaptic transmission of auditory signals.