Combined Retrograde Tracing Research Articles

The nociceptive inputs of the paraventricular hypothalamic nucleus in formalin stimulated mice

The paraventricular hypothalamic nucleus (PVH) is an important neuroendocrine center involved in pain regulation, but the nociceptive afferent routes for the nucleus are still unclear. We examined the profile of PVH receiving injurious information by a combination of retrograde tracing with Fluoro-Gold (FG) and FOS expression induced by formalin stimuli. The result showed that formalin injection induced significantly increased expression of FOS in the PVH, among which oxytocin containing neurons are one neuronal phenotype. Immunofluorescent staining of FG and FOS revealed that double labeled neurons were strikingly distributed in the area 2 of the cingulate cortex (Cg2), the lateral septal nucleus (LS), the periaqueductal gray (PAG), the posterior hypothalamic area (PH), and the lateral parabrachial nucleus (LPB). In the five regions, LPB had the biggest number and the highest ratio of FOS expression in FG labeled neurons, with main subnuclei distribution in the external, superior, dorsal, and central parts. Further immunofluorescent triple staining disclosed that about one third of FG and FOS double labeled neurons in the LPB were immunoreactive for calcitonin gene related peptide (CGRP). In conclusion, the present study demonstrates the nociceptive input profile of the PVH area under inflammatory pain and suggests that neurons in the LPB may play essential roles in transmitting noxious information to the PVH.

Recruitment of hippocampal and thalamic pathways to the central amygdala in the control of feeding behavior under novelty.

It is adaptive to restrict eating under uncertainty, such as during habituation to novel foods and unfamiliar environments. However, sustained restrictive eating can become maladaptive. Currently, the neural substrates of restrictive eating are poorly understood. Using a model of feeding avoidance under novelty, our recent study identified forebrain activation patterns and found evidence that the central nucleus of the amygdala (CEA) is a core integrating node. The current study analyzed the activity of CEA inputs in male and female rats to determine if specific pathways are recruited during feeding under novelty. Recruitment of direct inputs from the paraventricular nucleus of the thalamus (PVT), the infralimbic cortex (ILA), the agranular insular cortex (AI), the hippocampal ventral field CA1, and the bed nucleus of the stria terminals (BST) was assessed with combined retrograde tract tracing and Fos induction analysis. The study found that during consumption of a novel food in a novel environment, larger number of neurons within the PVTp and the CA1 that send monosynaptic inputs to the CEA were recruited compared to controls that consumed familiar food in a familiar environment. The ILA, AI, and BST inputs to the CEA were similarly recruited across conditions. There were no sex differences in activation of any of the pathways analyzed. These results suggest that the PVTp-CEA and CA1-CEA pathways underlie feeding inhibition during novelty and could be potential sites of malfunction in excessive food avoidance.

Neural pathways from the central nucleus of the amygdala to the paraventricular nucleus of the hypothalamus are involved in induction of yawning behavior due to emotional stress in rats

As yawning is often observed in stressful or emotional situations such as tension and anxiety, this suggests that yawning can be considered to be an emotional behavior. However, the neural mechanisms underlying emotion-induced yawning remain unclear. It is well known that the hypothalamic paraventricular nucleus (PVN) is the most important brain structure for induction of yawning behavior. We previously showed that induction of yawning involves the central nucleus of the amygdala (CeA), as well as the PVN. Therefore, emotion-induced yawning could potentially be induced through activation of the direct/indirect neural pathways from the CeA to the PVN. Our present study used a combination of retrograde tracing (injection of Fluoro-Gold (FG) into the PVN) and c-Fos immunohistochemistry to examine the neural pathways that evoke emotion-induced yawning. We additionally performed lesion experiments on the CeA using ibotenic acid, a neurotoxin, to determine whether the CeA is involved in the induction of emotion-induced yawning. Emotional stress by fear conditioning induced yawning behavior, and induced expression of double-labeled cells for c-Fos and FG in the bed nucleus of the stria terminalis (BNST), but not in the CeA. Furthermore, the CeA lesions caused by ibotenic acid abolished the induction of emotion-induced yawning. These results suggest that a neural pathway from the CeA to the PVN via the BNST may be primarily involved in the induction of emotion-induced yawning behavior.

Muscarinic M4 and M5 receptors in the ventral subiculum differentially modulate alcohol seeking versus consumption in male alcohol-preferring rats.

Muscarinic acetylcholine receptors mediate alcohol consumption and seeking in rats. While M4 and M5 receptors have recently been implicated to mediate these behaviours in the striatum, their role in other brain regions remain unknown. The ventral tegmental area (VTA) and ventral subiculum (vSub) both densely express M4 and M5 receptors and modulate alcohol-seeking, via their projections to the nucleus accumbens shell (AcbSh). In Indiana alcohol-preferring (iP) male rats, we examined Chrm4 (M4 ) and Chrm5 (M5 ) expression in the VTA and vSub following long-term alcohol consumption and abstinence using RT-qPCR. Using a combination of retrograde tracing and RNAscope, we examined the localisation of Chrm4 and Chrm5 on vSub cells that project to the AcbSh. Using selective allosteric modulators, we examined the functional role of M4 and M5 receptors within the vSub in alcohol consumption, context-induced alcohol-seeking, locomotor activity, and food/water consumption. Long-term alcohol and abstinence dysregulated the expression of genes for muscarinic receptors in the vSub, not in the VTA. Chrm4 was down-regulated following long-term alcohol and abstinence, while Chrm5 was up-regulated following long-term alcohol consumption. Consistent with these data, a positive allosteric modulator (VU0467154) of intra-vSub M4 receptors reduced context-induced alcohol-seeking, but not motivation for alcohol self-administration, while M5 receptor negative allosteric modulator (ML375) reduced initial motivation for alcohol self-administration, but not context-induced alcohol-seeking. Collectively, our data highlight alcohol-induced cholinergic dysregulation in the vSub and distinct roles for M4 and M5 receptor allosteric modulators to reduce alcohol consumption or seeking.

The CSF-Contacting Nucleus Receives Anatomical Inputs From the Cerebral Cortex: A Combination of Retrograde Tracing and 3D Reconstruction Study in Rat.

ObjectiveThis study aimed to investigate the direct monosynaptic projections from cortical functional regions to the cerebrospinal fluid (CSF)-contacting nucleus for understanding the functions of the CSF-contacting nucleus.MethodsThe Sprague–Dawley rats received cholera toxin B subunit (CB) injections into the CSF-contacting nucleus. After 7–10 days of survival time, the rats were perfused, and the whole brain and spinal cord were sliced under a freezing microtome at 40 μm. All sections were treated with the CB immunofluorescence reaction. The retrogradely labeled neurons in different cortical areas were revealed under a confocal microscope. The distribution features were further illustrated under 3D reconstruction.ResultsThe retrogradely labeled neurons were identified in the olfactory, orbital, cingulate, insula, retrosplenial, somatosensory, motor, visual, auditory, association, rhinal, and parietal cortical areas. A total of 12 functional areas and 34 functional subregions showed projections to the CSF-contacting nucleus in different cell intensities.ConclusionAccording to the connectivity patterns, we conclude that the CSF-contacting nucleus participates in cognition, emotion, pain, visceral activity, etc. The present study firstly reveals the cerebral cortex→CSF-contacting nucleus connections, which implies the multiple functions of this special nucleus in neural and body fluid regulations.

Anatomically and functionally distinct locus coeruleus efferents mediate opposing effects on anxiety-like behavior

The locus coeruleus (LC) is a critical node in the stress response, and its activation has been shown to promote hypervigilance and anxiety-like behavior. This noradrenergic nucleus has historically been considered homogeneous with highly divergent neurons that operate en masse to collectively affect central nervous system function and behavioral state. However, in recent years, LC has been identified as a heterogeneous structure whose neurons innervate discrete terminal fields and contribute to distinct aspects of behavior. We have previously shown that in late adolescent male rats, an acute traumatic stressor, simultaneous physical restraint and exposure to predator odor, preferentially induces c-Fos expression in a subset of dorsal LC neurons and persistently increases anxiety-like behavior. To investigate how these neurons respond to and contribute to the behavioral response to stress, we used a combination of retrograde tracing, whole-cell patch clamp electrophysiology, and chemogenetics. Here we show that LC neurons innervating the central nucleus of the amygdala (CeA) and medial prefrontal cortex (mPFC) undergo distinct electrophysiological changes in response to stressor exposure and have opposing roles in mediating anxiety-like behavior. While neurons innervating CeA become more excitable in response to stress and promote anxiety-like behavior, those innervating mPFC become less excitable and appear to promote exploration. These findings show that LC neurons innervating distinct terminal fields have unique physiological responses to particular stimuli. Furthermore, these observations advance the understanding of the LC as a complex and heterogeneous structure whose neurons maintain unique roles in various forms of behavior.

ASIC3 inhibition modulates inflammation-induced changes in the activity and sensitivity of Aδ and C fiber sensory neurons that innervate bone.

The Acid Sensing Ion Channel 3 (ASIC3) is a non-selective cation channel that is activated by acidification, and is known to have a role in regulating inflammatory pain. It has pro-algesic roles in a range of conditions that present with bone pain, but the mechanism for this has not yet been demonstrated. We aimed to determine if ASIC3 is expressed in Aδ and/or C fiber bone afferent neurons, and to explore its role in the activation and sensitization of bone afferent neurons after acute inflammation. A combination of retrograde tracing and immunohistochemistry was used to determine expression of ASIC3 in the soma of bone afferent neurons. A novel, in vivo, electrophysiological bone-nerve preparation was used to make recordings of the activity and sensitivity of bone afferent neurons in the presence of carrageenan-induced inflammation, with and without the selective ASIC3 inhibitor APET×2. A substantial proportion of bone afferent neurons express ASIC3, including unmyelinated (neurofilament poor) and small diameter myelinated (neurofilament rich) neurons that are likely to be C and Aδ nerve fibers respectively. Electrophysiological recordings revealed that application of APET×2 to the marrow cavity inhibited carrageenan-induced spontaneous activity of C and Aδ fiber bone afferent neurons. APET×2 also inhibited carrageenan-induced sensitization of Aδ and C fiber bone afferent neurons to mechanical stimulation, but had no effect on the sensitivity of bone afferent neurons in the absence of inflammation. This evidence supports a role for ASIC3 in the pathogenesis of pain associated with inflammation of the bone.

TRPV1 activation alters the function of Aδ and C fiber sensory neurons that innervate bone.

The Transient receptor potential cation channel subfamily V member 1 (TRPV1) is a non-selective cation channel that is activated by capsaicin, low pH and noxious heat. It has been suggested to have a pro-algesic role in a range of conditions that present with bone pain, but the mechanisms by which this occurs are not yet clear. In this study we aimed to determine if TRPV1 is expressed in Aδ and/or C fiber bone afferent neurons, and to explore its role in the activation and/or sensitization of bone afferent neurons to mechanical stimulation. A combination of retrograde tracing and immunohistochemistry was used to determine expression of TRPV1 in the soma of bone afferent neurons that innervate the rat tibial marrow cavity. A novel, in vivo, electrophysiological bone-nerve preparation, recently developed in our laboratory, was used to make recordings of the activity and sensitivity of bone afferent neurons in response to application of the TRPV1 agonist capsaicin to the marrow cavity. We found that a substantial proportion of bone afferent neurons express TRPV1. These include both small-diameter myelinated (neurofilament rich) and unmyelinated (neurofilament poor) neurons that are likely to be Aδ and C fiber neurons, respectively. Electrophysiological recordings revealed that application of capsaicin to the marrow cavity increased ongoing activity of C fiber, and to a lesser extent Aδ fiber, bone afferent neurons. Capsaicin also sensitized both Aδ and C fiber bone afferent neurons to mechanical stimulation. This evidence supports a role for TRPV1 in the pathogenesis of pain associated with bone pathology or disease.

Hypoxia activates a neuropeptidergic pathway from the paraventricular nucleus of the hypothalamus to the nucleus tractus solitarii.

The paraventricular nucleus of the hypothalamus (PVN) contributes to both autonomic and neuroendocrine function. PVN lesion or inhibition blunts cardiorespiratory responses to peripheral chemoreflex activation, suggesting that the PVN is required for full expression of these effects. However, the role of efferent projections to cardiorespiratory nuclei and the neurotransmitters/neuromodulators that are involved is unclear. The PVN sends dense projections to the nucleus tractus solitarii (nTS), a region that displays neuronal activation following hypoxia. We hypothesized that acute hypoxia activates nTS-projecting PVN neurons. Using a combination of retrograde tracing and immunohistochemistry, we determined whether hypoxia activates PVN neurons that project to the nTS and examined the phenotype of these neurons. Conscious rats underwent 2 h normoxia (21% O2, n = 5) or hypoxia (10% O2, n = 6). Hypoxia significantly increased Fos immunoreactivity in nTS-projecting neurons, primarily in the caudal PVN. The majority of activated nTS-projecting neurons contained corticotropin-releasing hormone (CRH). In the nTS, fibers expressing the CRH receptor corticotropin-releasing factor receptor 2 (CRFR2) were colocalized with oxytocin (OT) fibers and were closely associated with hypoxia-activated nTS neurons. A separate group of animals that received a microinjection of adeno-associated virus type 2-hSyn-green fluorescent protein (GFP) into the PVN exhibited GFP-expressing fibers in the nTS; a proportion of these fibers displayed OT immunoreactivity. Thus, nTS CRFR2s appear to be located on the fibers of PVN OT neurons that project to the nTS. Taken together, our findings suggest that PVN CRH projections to the nTS may modulate nTS neuronal activation, possibly via OTergic mechanisms, and thus contribute to chemoreflex cardiorespiratory responses.

Lateral hypothalamic Orexin-A-ergic projections to the arcuate nucleus modulate gastric function in vivo.

It has been well-known that hypothalamic orexigenic neuropeptides, orexin-A, and melanin-concentrating hormone (MCH), play important roles in regulation of gastric function. However, what neural pathway mediated by the two neuropeptides affects the gastric function remains unknown. In this study, by way of nucleic stimulation and extracellular recording of single unit electrophysiological properties, we found that electrically stimulating the lateral hypothalamic area (LH) or microinjection of orexin-A into the arcuate nucleus (ARC) excited most gastric distension-responsive neurons in the nuclei and enhanced the gastric function including motility, emptying, and acid secretion of conscious rats. The results indicated that LH-ARC orexin-A-ergic projections may exist and the orexin-A in the ARC affected afferent and efferent signal transmission between ARC and stomach. As expected, combination of retrograde tracing and immunohistochemistry showed that some orexin-A-ergic neurons projected from the LH to the ARC. In addition, microinjection of MCH and its receptor antagonist PMC-3881-PI into the ARC affected the role of orexin-A in the ARC, indicating a possible involvement of the MCH pathway in the orexin-A role. Our findings suggest that there was an orexin-A-ergic pathway between LH and ARC which participated in transmitting information between the central nuclei and the gastrointestinal tract and in regulating the gastric function of rats.

Locus coeruleus to basolateral amygdala noradrenergic projections promote anxiety-like behavior.

Increased tonic activity of locus coeruleus noradrenergic (LC-NE) neurons induces anxiety-like and aversive behavior. While some information is known about the afferent circuitry that endogenously drives this neural activity and behavior, the downstream receptors and anatomical projections that mediate these acute risk aversive behavioral states via the LC-NE system remain unresolved. Here we use a combination of retrograde tracing, fast-scan cyclic voltammetry, electrophysiology, and in vivo optogenetics with localized pharmacology to identify neural substrates downstream of increased tonic LC-NE activity in mice. We demonstrate that photostimulation of LC-NE fibers in the BLA evokes norepinephrine release in the basolateral amygdala (BLA), alters BLA neuronal activity, conditions aversion, and increases anxiety-like behavior. Additionally, we report that β-adrenergic receptors mediate the anxiety-like phenotype of increased NE release in the BLA. These studies begin to illustrate how the complex efferent system of the LC-NE system selectively mediates behavior through distinct receptor and projection-selective mechanisms.

Detailed Characterization of Sympathetic Chain Ganglia (SChG) Neurons Supplying the Skin of the Porcine Hindlimb.

It is generally known that in the skin sympathetic fibers innervate various dermal structures, including sweat glands, blood vessels, arrectores pilorum muscles and hair follicles. However, there is a lack of data about the distribution and chemical phenotyping of the sympathetic chain ganglia (SChG) neurons projecting to the skin of the pig, a model that is physiologically and anatomically very representative for humans. Thus, the present study was designed to establish the origin of the sympathetic fibers supplying the porcine skin of the hind leg, and the pattern(s) of putative co-incidence of dopamine-β-hydroxylase (DβH) with pituitary adenylate cyclase-activating polypeptide (PACAP), somatostatin (SOM), neuronal nitric oxide synthase, substance P, vasoactive intestinal peptide, neuropeptide Y (NPY), leu5-enkephalin and galanin (GAL) using combined retrograde tracing and double-labeling immunohistochemistry. The Fast Blue-positive neurons were found in the L2–S2 ganglia. Most of them were small-sized and contained DβH with PACAP, SOM, NPY or GAL. The findings of the present study provide a detailed description of the distribution and chemical coding of the SChG neurons projecting to the skin of the porcine hind leg. Such data may be the basis for further studies concerning the plasticity of these ganglia under experimental or pathological conditions.

Orexinergic fibers are in contact with Kölliker-Fuse nucleus neurons projecting to the respiration-related nuclei in the medulla oblongata and spinal cord of the rat

The neural pathways underlying the respiratory variation dependent on vigilance states remain unsettled. In the present study, we examined the orexinergic innervation of Kölliker-Fuse nucleus (KFN) neurons sending their axons to the rostral ventral respiratory group (rVRG) and phrenic nucleus (PhN) as well as to the hypoglossal nucleus (HGN) by using a combined retrograde tracing and immunohistochemistry. After injection of cholera toxin B subunit (CTb) into the KFN, CTb-labeled neurons that are also immunoreactive for orexin (ORX) were found prominently in the perifornical and medial regions and additionally in the lateral region of the hypothalamic ORX field. After injection of fluorogold (FG) into the rVRG, PhN or HGN, we found an overlapping distribution of ORX-immunoreactive axon terminals and FG-labeled neurons in the KFN. Within the neuropil of the KFN, asymmetrical synaptic contacts were made between these terminals and neurons. We further demonstrated that many neurons labeled with FG injected into the rVRG, PhN, or HGN are immunoreactive for ORX receptor 2. Present data suggest that rVRG-, PhN- and HGN-projecting KFN neurons may be under the excitatory influence of the ORXergic neurons for the state-dependent regulation of respiration.

Immunohistochemical characteristics and distribution of neurons in the intramural ganglia supplying the urinary bladder in the male pig

This study investigated the distribution and chemical coding of neurons in intramural ganglia of the urinary bladder trigone (UBT-IG) and cervix (UBC-IG) in the male pig using combined retrograde tracing and double-labelling immunohistochemistry. Additionally, immunoblotting was used to confirm the presence of marker enzymes for main populations of autonomic neurons. Retrograde fluorescent tracer Fast Blue (FB) was injected into the wall of both the left and right side of the bladder trigone, cervix and apex during laparotomy performed under thiopental anaesthesia. Twelve tm-thick cryostat sections were processed for double-labelling immunofluorescence with antibodies against tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), neuropeptide Y (NPY), somatostatin (SOM), galanin (GAL), vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), calcitonin gene-related peptide (CGRP), substance P (SP) and vesicular acetylcholine transporter (VAChT). UBT-IG and UBC-IG neurons in both parts of the organ formed characteristic clusters (from few to tens of neuronal cells) found under visceral peritoneum or in the outer muscular layer. Immunohistochemistry revealed several subpopulations in UBT-IG and UBC-IG neurons, namely noradrenergic (ca. 76% and 76%), cholinergic (ca. 22% and 20%), non-adrenergic/non-cholinergic nerve cells (ca. 1.5% and 3.8%), NPY- (ca. 66% and 58%), SOM- (ca. 39% and 39 %), VIP- (ca. 5% and 0%) and NOS- immunoreactive (IR) (ca. 1.5% and 3.8%), respectively. Immunoblotting using antibodies to TH and VAChT showed the presence of studied proteins as revealed by the presence of protein bands of the correct molecular weight. This study has revealed a relatively large population of differently coded UBT- and UBC- IG neurons, which constitute an important element of the complex neuroendocrine system involved in the regulation of the male urogenital organs function.

Immunohistochemical Characteristics and Distribution of Neurons in the Paravertebral, Prevertebral and Pelvic Ganglia Supplying the Urinary Bladder in the Male Pig

The distribution and chemical coding of neurons supplying urinary bladder in the male pig were studied in the sympathetic chain ganglia, inferior mesenteric ganglia and anterior pelvic ganglia. The combined retrograde tracing and immunohistochemistry for tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), neuropeptide Y (NPY), somatostatin(SOM), galanin (GAL), vasoactive intestinal polypeptide(VIP), nitric oxide synthase (NOS), calcitonin gene related peptide (CGRP), substance P (SP), choline acetyltransferase (ChAT) and vesicular acetylcholine transporter(VAChT) were applied in the experiment. Bladder projecting neurons were found in all the ganglia studied. The majority of sympathetic ganglia neurons (inferior mesenteric ganglia and sympathetic chain ganglia) expressed immunoreactivity(IR) to DBH. In sympathetic chain ganglia these neurons simultaneously expressed NPY, GAL or VAChT,while in inferior mesenteric ganglia they contained NPY, SOM and/or GAL. A small number of these bladder projecting neurons was VAChT-IR and some contained NPY. In the pelvic ganglia bladder-projecting neurons formed two populations: DBH- and VAChT-IR. Some of DBH-IR neurons contained IR to NPY, SOM or GAL, while VAChTIR neurons were NPY-, SOM- or NOS-IR. The results indicate that sympathetic ganglia contain mainly adrenergic neurons,while pelvic ganglia contain both adrenergic and cholinergic neurons. All these neurons contain typical combinations of neuropeptides.

Intrapancreatic ganglia neurons receive projection fibers from melanocortin-4 receptor-expressing neurons in the dorsal motor nucleus of the vagus nerve of the mouse

Melanocortin-4 receptor (MC4R)-expressing neurons are widely distributed in the central nervous system and play a crucial role in a variety of physiological functions including energy and glucose/insulin homeostasis. However, their neural pathways remain to be elucidated. In the present study, we examined a possible pathway from MC4R-expressing neurons in the dorsal motor nucleus of the vagus nerve (DMV) to the intrapancreatic ganglia using transgenic mice that express green fluorescent protein (GFP) under the control of the MC4R-promoter. Using immunofluorescence labeling, we demonstrated that GFP-immunoreactive (ir) nerve fibers were distributed in the intrapancreatic ganglia closely associated with the islets as well as among the acini. These GFP-ir fibers with bouton-like varicosities were frequently observed to surround ganglion cells immunoreactive for vasoactive intestinal polypeptide, a marker for postganglionic parasympathetic neurons. Using the pre-embedding immunoperoxidase method, we clearly showed that GFP-ir terminals formed synapses predominantly with dendrites and additionally with somata of the ganglion cells. Moreover, bilateral subdiaphragmatic vagotomy caused a marked loss of GFP immunoreactivity in the pancreas. Using a combination of retrograde tracing and immunohistochemistry, we finally demonstrated that nearly half of the pancreas-projecting DMV neurons were immunoreactive for GFP. These results suggest that MC4R-expressing DMV neurons may participate in the regulation of glucose/insulin homeostasis through their projections to the intrapancreatic ganglia.

Central stress-integrative circuits: forebrain glutamatergic and GABAergic projections to the dorsomedial hypothalamus, medial preoptic area, and bed nucleus of the stria terminalis

Central regulation of hypothalamo-pituitary-adrenocortical (HPA) axis stress responses is mediated by a relatively circumscribed group of projections to the paraventricular hypothalamus (PVN). The dorsomedial hypothalamus (DMH), medial preoptic area (mPOA), and bed nucleus of the stria terminalis (BST) provide direct, predominantly inhibitory, innervation of the PVN. These PVN-projecting neurons are controlled by descending information from limbic forebrain structures, including the prefrontal cortex, amygdala, hippocampus, and septum. The neurochemical phenotype of limbic circuits targeting PVN relays has not been systematically analyzed. The current study combined retrograde tracing and immunohistochemistry/in situ hybridization to identify the specific sites of glutamatergic and GABAergic inputs to the DMH, mPOA, and BST. Following Fluoro-gold (FG) injections in the DMH, retrogradely labeled cells co-localized with vesicular glutamate transporter mRNA in the prefrontal cortex, ventral hippocampus, and paraventricular thalamus. Co-localization of FG and glutamic acid decarboxylase mRNA was present throughout the central and medial amygdaloid nuclei and septal area. In addition, the mPOA received predominantly GABAergic input from the septum, amygdala, and BST. The BST received glutamatergic projections from the hippocampus and basomedial amygdala, whereas, GABAergic inputs arose from central and medial amygdaloid nuclei. Thus, discrete sets of neurons in the hypothalamus and BST are positioned to summate limbic inputs into PVN regulation and may play a role in HPA dysfunction and stress-related illness.

A Cholinergic Mechanism for Reward Timing within Primary Visual Cortex

Neurons in rodent primary visual cortex (V1) relate operantly conditioned stimulus-reward intervals with modulated patterns of spiking output, but little is known about the locus or mechanism of this plasticity. Here we show that cholinergic basal forebrain projections to V1 are necessary for the neural acquisition, but not the expression, of reward timing in the visual cortex of awake, behaving animals. We then mimic reward timing invitro by pairing white matter stimulation with muscarinic receptor activation at a fixed interval and show that this protocol results in the prolongation of electrically evoked spike train durations out to the conditioned interval. Together, these data suggest that V1 possesses the circuitry and plasticity to support reward time prediction learning and the cholinergic system serves as an important reinforcement signal which, invivo, conveys to the cortex the outcome of behavior.

Delayed Dominant-Negative TNF Gene Therapy Halts Progressive Loss of Nigral Dopaminergic Neurons in a Rat Model of Parkinson's Disease

Parkinson's disease (PD) is a progressive neurodegenerative disorder typified by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). Recent evidence indicates that neuroinflammation may play a critical role in the pathogenesis of PD, particularly tumor necrosis factor (TNF). We have previously shown that soluble TNF (solTNF) is required to mediate robust degeneration induced by 6-hydroxydopamine (6-OHDA) or lipopolysaccharide. What remains unknown is whether TNF inhibition can attenuate the delayed and progressive phase of neurodegeneration. To test this, rats were injected in the SNpc with lentivirus encoding dominant-negative TNF (lenti-DN-TNF) 2 weeks after receiving a 6-OHDA lesion. Remarkably, when examined 5 weeks after the initial 6-OHDA lesion, no further loss of nigral DA neurons was observed. Lenti-DN-TNF also attenuated microglial activation. Together, these data suggest that TNF is likely a critical mediator of nigral DA neuron death during the delayed and progressive phase of neurodegeneration, and that microglia may be the principal cell type involved. These promising findings provide compelling reasons to perform DN-TNF gene transfer studies in nonhuman primates with the long-term goal of using it in the clinic to prevent the delayed and progressive degeneration of DA neurons that gives rise to motor symptoms in PD.

The Highly Conserved Gonadotropin-Releasing Hormone-2 Form Acts as a Melatonin-Releasing Factor in the Pineal of a Teleost Fish, the European Sea Bass Dicentrarchus labrax

With the exception of modern mammals, most vertebrate species possess two GnRH genes, GnRH-1 and GnRH-2. In addition, in many teleost fish, there is a third gene called GnRH-3. If the main function of GnRH-1 is unambiguously to stimulate gonadotropin release, the other two GnRH forms still lack clear functions. This is particularly true for the highly conserved GnRH-2 that encodes chicken GnRH-II. This GnRH variant is consistently expressed in neurons of the dorsal synencephalon in most vertebrate groups but still has no clear functions supported by anatomical, pharmacological, and physiological data. In this study performed on a perciform fish, the European sea bass, we show for the first time that the pineal organ receives GnRH-2-immunoreactive fibers originating from the synencephalic GnRH-2 neurons. This was shown through a combination of retrograde tracing and immunohistochemistry, using highly specific antibodies. Supporting the presence of GnRH-2 functional targets, RT-PCR data together with the in situ hybridization studies showed that the sea bass pineal gland strongly expressed a GnRH receptor (dlGnRHR-II-2b) with clear selectivity for GnRH-2 and, to a lesser extent, the dlGnRHR-II-1a subtype. Finally, in vitro and in vivo experiments demonstrate stimulatory effects of GnRH-2 on nocturnal melatonin secretion by the sea bass pineal organ. Altogether, these data provide, for the first time in a vertebrate species, converging evidence supporting a role of GnRH-2 in the modulation of fish pineal functions.