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.

Orexinergic neurons are involved in the chemosensory control of breathing during the dark phase in a Parkinson's disease model.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic neurons in the substantia nigra compacta (SNpc) and the only risk factor is aging. We showed that in 6-hydroxydopamine (6-OHDA)-model of PD there is a reduction in the neuronal profile within the brainstem ventral respiratory column with a decrease in the hypercapnic ventilatory response. Here we tested the involvement of orexin cells from the lateral hypothalamus/perifornical area (LH/PeF) on breathing in a 6-OHDA PD model. In this model of PD, there is a reduction in the total number of orexinergic neurons and in the number of orexinergic neurons that project to the RTN, without changing the number of CO2-activated orexinergic neurons during the dark phase. The ventilation at rest and in response to hypercapnia (7% CO2) was assessed in animals that received 6-OHDA or vehicle injections into the striatum and saporin anti-Orexin-B or IgG saporin into the LH/PeF during the sleep and awake states. The experiments showed a reduction of respiratory frequency (fR) at rest during the light phase in PD animals only during sleep. During the dark phase, there was an impaired fR response to hypercapnia in PD animals with depletion of orexinergic neurons in awake and sleeping rats. In conclusion, the degeneration of orexinergic neurons in this model of PD can be related to impaired chemoreceptor function in the dark phase.

The Cerebello-Hypothalamic and Hypothalamo-Cerebellar Pathways via Superior and Middle Cerebellar Peduncle in the Rat.

The connections between the cerebellum and the hypothalamus have been well documented. However, the specific cerebellar peduncle through which the hypothalamo-cerebellar and cerebello-hypothalamic connections pass has not been demonstrated. The present study aims to define the specific cerebellar peduncle through which connects the cerebellum to specific hypothalamic nuclei. Seventeen male albino rats received 20-50-nl pressure injections of either Fluoro-Gold (FG) or biotinylated dextran amine (BDA) tracer into the superior (SCP), middle (MCP), and inferior (ICP) cerebellar peduncle. Following 7-10 days of survival period, the animals were processed according to the appropriate protocol for the two tracers used. Labeled cells and axons were documented using light or fluorescence microscopy. The present study showed connections between the hypothalamus and the cerebellum via both the SCP and the MCP but not the ICP. The hypothalamo-cerebellar connections via the SCP were from the lateral, dorsomedial, paraventricular, and posterior hypothalamic nuclei, and cerebello-hypothalamic connections were to the preoptic and lateral hypothalamic nuclei. The hypothalamo-cerebellar connections via the MCP were from the lateral, dorsomedial, ventromedial, and mammillary hypothalamic nuclei; and cerebello-hypothalamic connections were to the posterior, arcuate, and ventromedial hypothalamic nuclei. The hypothlamo-cerebellar connections were denser compared to the cerebello-hypothlamic connections via both the SCP and the MCP. The connection between the cerebellum and the hypothalamus was more prominent via the SCP than MCP. Both the hypothlamo-cerebellar and cerebello-hypothalamic connections were bilateral, with ipsilateral preponderance. Reciprocal connections were with the lateral hypothalamic nucleus via the SCP and the ventromedial nucleus via the MCP were observed. Cerebellum takes part in the higher order brain functions via its extensive connections. The knowledge of hypothalamo-cerebellar and cerebello-hypothalamic connections conveyed within the SCP and MCP can be important for the lesions involving the MCP and SCP. These connections can also change the conceptual architecture of the cerebellar circuitry and deepen current understanding.

VGLUT1 or VGLUT2 mRNA-positive neurons in spinal trigeminal nucleus provide collateral projections to both the thalamus and the parabrachial nucleus in rats.

The trigemino-thalamic (T-T) and trigemino-parabrachial (T-P) pathways are strongly implicated in the sensory-discriminative and affective/emotional aspects of orofacial pain, respectively. These T-T and T-P projection fibers originate from the spinal trigeminal nucleus (Vsp). We previously determined that many vesicular glutamate transporter (VGLUT1 and/or VGLUT2) mRNA-positive neurons were distributed in the Vsp of the adult rat, and most of these neurons sent their axons to the thalamus or cerebellum. However, whether VGLUT1 or VGLUT2 mRNA-positive projection neurons exist that send their axons to both the thalamus and the parabrachial nucleus (PBN) has not been reported. Thus, in the present study, dual retrograde tract tracing was used in combination with fluorescence in situ hybridization (FISH) for VGLUT1 or VGLUT2 mRNA to identify the existence of VGLUT1 or VGLUT2 mRNA neurons that send collateral projections to both the thalamus and the PBN. Neurons in the Vsp that send collateral projections to both the thalamus and the PBN were mainly VGLUT2 mRNA-positive, with a proportion of 90.3%, 93.0% and 85.4% in the oral (Vo), interpolar (Vi) and caudal (Vc) subnucleus of the Vsp, respectively. Moreover, approximately 34.0% of the collateral projection neurons in the Vc showed Fos immunopositivity after injection of formalin into the lip, and parts of calcitonin gene-related peptide (CGRP)-immunopositive axonal varicosities were in direct contact with the Vc collateral projection neurons. These results indicate that most collateral projection neurons in the Vsp, particularly in the Vc, which express mainly VGLUT2, may relay orofacial nociceptive information directly to the thalamus and PBN via axon collaterals.

Direct and indirect nigrofugal projections to the nucleus reticularis pontis caudalis mediate in the motor execution of the acoustic startle reflex.

The acoustic startle reflex (ASR) is a short and intense defensive reaction in response to a loud and unexpected acoustic stimulus. In the rat, a primary startle pathway encompasses three serially connected central structures: the cochlear root neurons, the giant neurons of the nucleus reticularis pontis caudalis (PnC), and the spinal motoneurons. As a sensorimotor interface, the PnC has a central role in the ASR circuitry, especially the integration of different sensory stimuli and brain states into initiation of motor responses. Since the basal ganglia circuits control movement and action selection, we hypothesize that their output via the substantia nigra (SN) may interplay with the ASR primary circuit by providing inputs to PnC. Moreover, the pedunculopontine tegmental nucleus (PPTg) has been proposed as a functional and neural extension of the SN, so it is another goal of this study to describe possible anatomical connections from the PPTg to PnC. Here, we made 6-OHDA neurotoxic lesions of the SN pars compacta (SNc) and submitted the rats to a custom-built ASR measurement session to assess amplitude and latency of motor responses. We found that following lesion of the SNc, ASR amplitude decreased and latency increased compared to those values from the sham-surgery and control groups. The number of dopamine neurons remaining in the SNc after lesion was also estimated using a stereological approach, and it correlated with our behavioral results. Moreover, we employed neural tract-tracing techniques to highlight direct projections from the SN to PnC, and indirect projections through the PPTg. Finally, we also measured levels of excitatory amino acid neurotransmitters in the PnC following lesion of the SN, and found that they change following an ipsi/contralateral pattern. Taken together, our results identify nigrofugal efferents onto the primary ASR circuit that may modulate motor responses.

Early sensory experience influences the development of multisensory thalamocortical and intracortical connections of primary sensory cortices.

The nervous system integrates information from multiple senses. This multisensory integration already occurs in primary sensory cortices via direct thalamocortical and corticocortical connections across modalities. In humans, sensory loss from birth results in functional recruitment of the deprived cortical territory by the spared senses but the underlying circuit changes are not well known. Using tracer injections into primary auditory, somatosensory, and visual cortex within the first postnatal month of life in a rodent model (Mongolian gerbil) we show that multisensory thalamocortical connections emerge before corticocortical connections but mostly disappear during development. Early auditory, somatosensory, or visual deprivation increases multisensory connections via axonal reorganization processes mediated by non-lemniscal thalamic nuclei and the primary areas themselves. Functional single-photon emission computed tomography of regional cerebral blood flow reveals altered stimulus-induced activity and higher functional connectivity specifically between primary areas in deprived animals. Together, we show that intracortical multisensory connections are formed as a consequence of sensory-driven multisensory thalamocortical activity and that spared senses functionally recruit deprived cortical areas by an altered development of sensory thalamocortical and corticocortical connections. The functional-anatomical changes after early sensory deprivation have translational implications for the therapy of developmental hearing loss, blindness, and sensory paralysis and might also underlie developmental synesthesia.

Neuronal aromatase expression in pain processing regions of the medullary and spinal cord dorsal horn.

In both acute and chronic pain conditions, women tend to be more sensitive than men. This sex difference may be regulated by estrogens, such as estradiol, that are synthesized in the spinal cord and brainstem and act locally to influence pain processing. To identify a potential cellular source of local estrogen, here we examined the expression of aromatase, the enzyme that catalyzes the conversion of testosterone to estradiol. Our studies focused on primary afferent neurons and on their central targets in the spinal cord and medulla as well as in the nucleus of the solitary tract, the target of nodose ganglion-derived visceral afferents. Immunohistochemical staining in an aromatase reporter mouse revealed that many neurons in laminae I and V of the spinal cord dorsal horn and caudal spinal trigeminal nucleus and in the nucleus of the solitary tract express aromatase. The great majority of these cells also express inhibitory interneuron markers. We did not find sex differences in aromatase expression and neither the pattern nor the number of neurons changed in a sciatic nerve transection model of neuropathic pain or in the Complete Freund's adjuvant model of inflammatory pain. A few aromatase neurons express Fos after cheek injection of capsaicin, formalin, or chloroquine. In total, given their location, these aromatase neurons are poised to engage nociceptive circuits, whether it is through local estrogen synthesis or inhibitory neurotransmitter release.

Effects of Taxol on Regeneration in a Rat Sciatic Nerve Transection Model.

Recent studies describe taxol as a candidate treatment for promoting central nerve regeneration. However, taxol has serious side effects including peripheral neurotoxicity, and little information is known about the effect of taxol on peripheral nerve regeneration. We investigated the effects of taxol on regeneration in a rat sciatic nerve transection model. Rats were divided into four groups (n = 10): normal saline (i.p.) as the control, Cremophor EL vehicle, and 2 or 6 mg/kg of taxol in the Cremophor EL solution (four times in day-2, 4, 6, and 8), respectively. We evaluated neuronal electrophysiology, animal behaviour, neuronal connectivity, macrophage infiltration, location and expression levels of calcitonin gene-related peptide (CGRP), and expression levels of both nerve growth factors and immunoregulatory factors. In the high-dose taxol group (6 mg/kg), neuronal electrophysiological function was significantly impaired. Licking latencies were significantly changed while motor coordination was unaffected. Neuronal connectivity, macrophage density, and expression levels of CGRP was dramatically reduced. Expression levels of nerve growth factors and immunoregulatory factors was also reduced, while it was increased in the low-dose taxol group (2 mg/kg). These results indicate that taxol can modulate local inflammatory conditions, impair nerve regeneration, and impede recovery of a severe peripheral nerve injury.

Physiology and anatomy of neurons in the medial superior olive of the mouse.

In mammals with good low-frequency hearing, the medial superior olive (MSO) computes sound location by comparing differences in the arrival time of a sound at each ear, called interaural time disparities (ITDs). Low-frequency sounds are not reflected by the head, and therefore level differences and spectral cues are minimal or absent, leaving ITDs as the only cue for sound localization. Although mammals with high-frequency hearing and small heads (e.g., bats, mice) barely experience ITDs, the MSO is still present in these animals. Yet, aside from studies in specialized bats, in which the MSO appears to serve functions other than ITD processing, it has not been studied in small mammals that do not hear low frequencies. Here we describe neurons in the mouse brain stem that share prominent anatomical, morphological, and physiological properties with the MSO in species known to use ITDs for sound localization. However, these neurons also deviate in some important aspects from the typical MSO, including a less refined arrangement of cell bodies, dendrites, and synaptic inputs. In vitro, the vast majority of neurons exhibited a single, onset action potential in response to suprathreshold depolarization. This spiking pattern is typical of MSO neurons in other species and is generated from a complement of Kv1, Kv3, and IH currents. In vivo, mouse MSO neurons show bilateral excitatory and inhibitory tuning as well as an improvement in temporal acuity of spiking during bilateral acoustic stimulation. The combination of classical MSO features like those observed in gerbils with more unique features similar to those observed in bats and opossums make the mouse MSO an interesting model for exploiting genetic tools to test hypotheses about the molecular mechanisms and evolution of ITD processing.

Thalamic and Entorhinal Network Activity Differently Modulates the Functional Development of Prefrontal-Hippocampal Interactions.

Precise information flow during mnemonic and executive tasks requires the coactivation of adult prefrontal and hippocampal networks in oscillatory rhythms. This interplay emerges early in life, most likely as an anticipatory template of later cognitive performance. At neonatal age, hippocampal theta bursts drive the generation of prefrontal theta-gamma oscillations. In the absence of direct reciprocal interactions, the question arises of which feedback mechanisms control the early entrainment of prefrontal-hippocampal networks. Here, we demonstrate that prefrontal-hippocampal activity couples with discontinuous theta oscillations and neuronal firing in both lateral entorhinal cortex and ventral midline thalamic nuclei of neonatal rats. However, these two brain areas have different contributions to the neonatal long-range communication. The entorhinal cortex mainly modulates the hippocampal activity via direct axonal projections. In contrast, thalamic theta bursts are controlled by the prefrontal cortex via mutual projections and contribute to hippocampal activity. Thus, the neonatal prefrontal cortex modulates the level of hippocampal activation by directed interactions with the ventral midline thalamus. Similar to the adult task-related communication, theta-band activity ensures the feedback control of long-range coupling in the developing brain. SIGNIFICANCE STATEMENT: Memories are encoded by finely tuned interactions within large-scale neuronal networks. This cognitive performance is not inherited, but progressively matures in relationship with the establishment of long-range coupling in the immature brain. The hippocampus initiates and unidirectionally drives the oscillatory entrainment of neonatal prefrontal cortex, yet feedback interactions that precisely control this early communication are still unresolved. Here, we identified distinct roles of entorhinal cortex and ventral midline thalamus for the functional development of prefrontal-hippocampal interactions. While entorhinal oscillations modulate the hippocampal activity by timing the neuronal firing via monosynaptic afferents, thalamic nuclei act as a relay station routing prefrontal activation back to hippocampus. Understanding the mechanisms of network maturation represents the prerequisite for assessing circuit dysfunction in neurodevelopmental disorders.

Implantation of Miniosmotic Pumps and Delivery of Tract Tracers to Study Brain Reorganization in Pathophysiological Conditions.

Pharmacological treatment in animal models of cerebral disease imposes the problem of repeated injection protocols that may induce stress in animals and result in impermanent tissue levels of the drug. Additionally, drug delivery to the brain is delicate due to the blood brain barrier (BBB), thus significantly reducing intracerebral concentrations of selective drugs after systemic administration. Therefore, a system that allows both constant drug delivery without peak levels and circumvention of the BBB is in order to achieve sufficiently high intracerebral concentrations of drugs that are impermeable to the BBB. In this context, miniosmotic pumps represent an ideal system for constant drug delivery at a fixed known rate that eludes the problem of daily injection stress in animals and that may also be used for direct brain delivery of drugs. Here, we describe a method for miniosmotic pump implantation and post operatory care that should be given to animals in order to successfully apply this technique. We embed the aforementioned experimental paradigm in standard procedures that are used for studying neuroplasticity within the brain of C57BL6 mice. Thus, we exposed animals to 30 min brain infarct and implanted with miniosmotic pumps connected to the skull via a cannula in order to deliver a pro-plasticity drug. Behavioral testing was done during 30 days of treatment. After removal the animals received injections of anterograde tract tracers to analyze neuronal plasticity in the chronic phase of recovery. Results indicated that neuroprotection by the delivered drug was accompanied with increase in motor fibers crossing the midline of the brain at target structures. The results affirm the value of these techniques for drug administration and brain plasticity studies in modern neuroscience.

Estradiol Rapidly Attenuates ORL-1 Receptor-Mediated Inhibition of Proopiomelanocortin Neurons via Gq-Coupled, Membrane-Initiated Signaling.

Estradiol rapidly regulates the activity of arcuate nucleus (ARH) proopiomelanocortin (POMC) neurons that project to the medial preoptic nucleus (MPN) to regulate lordosis. Orphanin FQ/nociceptin (OFQ/N) acts via opioid receptor-like (ORL)-1 receptors to inhibit these POMC neurons. Therefore, we tested the hypothesis that estradiol excites POMC neurons by rapidly attenuating inhibitory ORL-1 signaling in these cells. Hypothalamic slices through the ARH were prepared from ovariectomized rats injected with Fluorogold into the MPN. Electrophysiological recordings were generated in ARH neurons held at or near -60 mV, and neuronal phenotype was determined post hoc by immunohistofluorescence. OFQ/N application induced robust outward currents and hyperpolarizations via G protein-gated, inwardly rectifying K+ (GIRK) channels that were attenuated by pretreatment with either 17-ß estradiol (E2) or E2 conjugated to bovine serum albumin. This was blocked by the estrogen receptor (ER) antagonist ICI 182,780 and mimicked by the Gq-coupled membrane ER (Gq-mER) ligand STX and the ERα agonist PPT. Inhibiting phosphatidylinositol-3-kinase (PI3K) blocked the estrogenic attenuation of ORL-1/GIRK currents. Antagonizing either phospholipase C (PLC), protein kinase C (PKC), protein kinase A (PKA) or neuronal nitric oxide synthase (nNOS) also abrogated E2 inhibition of ORL-1/GIRK currents, whereas activation of PKC, PKA, protein kinase B (Akt) and nNOS substrate L-arginine all attenuated the OFQ/N response. This was observed in 92 MPN-projecting, POMC-positive ARH neurons. Thus, ORL-1 receptor-mediated inhibition of POMC neurons is rapidly and negatively modulated by E2, an effect which is stereoselective and membrane initiated via Gq-mER and ERα activation that signals through PLC, PKC, PKA, PI3K and nNOS.

Anatomical evidence of pruriceptive trigeminothalamic and trigeminoparabrachial projection neurons in mice.

Itch is relayed to higher centers by projection neurons in the spinal and medullary dorsal horn. We employed a double-label method to map the ascending projections of pruriceptive and nociceptive trigeminal and spinal neurons. The retrograde tracer fluorogold (FG) was stereotaxically injected into the right thalamus or lateral parabrachial area (LPb) in mice. Seven days later, mice received intradermal (id) microinjection of histamine, chloroquine, capsaicin, or vehicle into the left cheek. Histamine, chloroquine, and capsaicin intradermally elicited similar distributions of Fos-positive neurons in the medial aspect of the superficial medullary and spinal dorsal horn from the trigeminal subnucleus caudalis to C2. Among neurons retrogradely labeled from the thalamus, 43%, 8%, and 22% were Fos-positive following id histamine, chloroquine, or capsaicin. Among the Fos-positive neurons following pruritic or capsaicin stimuli, ∼1-2% were retrogradely labeled with FG. Trigeminoparabrachial projection neurons exhibited a higher incidence of double labeling in the superficial dorsal horn. Among the neurons retrogradely labeled from LPb, 36%, 29%, and 33% were Fos positive following id injection of histamine, chloroquine, and capsaicin, respectively. Among Fos-positive neurons elicited by id histamine, chloroquine, and capsaicin, respectively, 3.7%, 4.3%, and 4.1% were retrogradely labeled from LPb. The present results indicate that, overall, relatively small subpopulations of pruriceptive and/or nociceptive neurons innervating the cheek project to thalamus or LPb. These results imply that the vast majority of pruritogen- and algogen-responsive spinal neurons are likely to function as interneurons relaying information to projection neurons and/or participating in segmental nocifensive circuits.

Nonspecific labeling limits the utility of Cre-Lox bred CST-YFP mice for studies of corticospinal tract regeneration.

Studies of axon regeneration in the spinal cord often assess regeneration of the corticospinal tract (CST). Emx1-Cre x Thy1-STOP-YFP mice have been reported to have yellow fluorescent protein (YFP) selectively expressed in forebrain neurons leading to genetic labeling of CST axons in the spinal cord, and it was suggested that these CST-YFP mice would be useful for studies of CST regeneration. Because regeneration past a lesion may involve only a few axons, the presence of labeled non-CST axons compromises interpretation. We show here that in CST-YFP mice, some YFP-labeled axons are not from the CST. Specifically, YFP-labeled axons are present in regions beyond those with anterogradely labeled CST axons, most YFP-labeled axons beyond established CST locations do not undergo Wallerian degeneration following a large lesion of the sensorimotor cortex, some rubrospinal and reticulospinal neurons are labeled with YFP, and some YFP-labeled cells in the spinal gray matter have YFP-labeled projections into the spinal cord white matter. We further demonstrate that the density of YFP-labeled axon arbors hinders tracing of single axons to their point of origin in the main descending tracts. In light of recent advances in 3D imaging for visualizing axons in unsectioned blocks of spinal cord, we also assessed CST-YFP mice for 3D imaging and found that YFP fluorescence in CST-YFP mice is faint for clearing-based 3D imaging in comparison with fluorescence in Thy1-YFP-H mice and fluorescence of mini-ruby biotinylated dextran amine (BDA). Overall, the nonspecific and faint YFP labeling in CST-YFP mice limits their utility for assessments of CST axon regeneration.

Melanin-concentrating hormone inputs to the nucleus accumbens originate from distinct hypothalamic sources and are apposed to GABAergic and cholinergic cells in the Long-Evans rat brain.

Melanin-concentrating hormone [MCH] is a neuropeptide that modulates several behaviors, such as feeding and reward. Because the hedonic and rewarding features of a food also influence feeding behavior, the nucleus accumbens [Acb] has been highlighted as a key area integrating these roles. Functional data confirm that MCH acts on a subdivision of the Acb; however, considering the importance of finding anatomical and neurochemical data that correlate the previously demonstrated function of MCH, we delineated this investigation based on the following points: (1) Is there a pattern of innervation by MCH fibers regarding the subregions within the Acb? (2) Specifically, which hypothalamic nuclei synthesize MCH and innervate the Acb? (3) Finally, what are the neurochemical identities of the accumbal neurons innervated by MCH inputs? We examined the MCH immunoreactivity [MCH-ir] in the Acb in rat brains using the peroxidase technique. Additionally, after injecting retrograde neuronal tracer [Fluoro-Gold® - FG®] into subdivisions of the Acb [shell or core], we mapped single- or double-labeled cells. Moreover, using a double immunoperoxidase protocol, we investigated the MCH-ir fibers for gamma-aminobutyric acid [GABA]-ir and choline acetyltransferase [ChAT]-ir cells in the shell subdivision of the Acb [AcbSh]. We found that the MCH-ir fibers preferentially innervate the medial AcbSh, particularly the septal pole. This innervation originated from the incerto-hypothalamic area [IHy], internuclear area, lateral hypothalamic area, perifornical area, periventricular nucleus and posterior hypothalamus. Moreover, the IHy has the highest relationship between double/single retrogradely labeled cells [n=5.33±0.66/16±0.93, i.e. 33.33%] in the whole hypothalamus. Furthermore, our data suggest that MCH-ir fibers are in apposition to GABAergic and cholinergic cells in the AcbSh. Therefore, we provide anatomical support to the ongoing functional studies investigating the relation among the hypothalamus, MCH transmission into the Acb and the involvement of known neuronal phenotypes within the AcbSh.

Musculotopic organization of the motor neurons supplying the mouse hindlimb muscles: a quantitative study using Fluoro-Gold retrograde tracing.

We have mapped the motor neurons (MNs) supplying the major hindlimb muscles of transgenic (C57/BL6J-ChAT-EGFP) and wild-type (C57/BL6J) mice. The fluorescent retrograde tracer Fluoro-Gold was injected into 19 hindlimb muscles. Consecutive transverse spinal cord sections were harvested, the MNs counted, and the MN columns reconstructed in 3D. Three longitudinal MN columns were identified. The dorsolateral column extends from L4 to L6 and consists of MNs innervating the crural muscles and the foot. The ventrolateral column extends from L1 to L6 and accommodates MNs supplying the iliopsoas, gluteal, and quadriceps femoris muscles. The middle part of the ventral horn hosts the central MN column, which extends between L2 and L6 and consists of MNs for the thigh adductor, hamstring, and quadratus femoris muscles. Within these longitudinal columns, the arrangement of the different MN groups reflects their somatotopic organization. MNs innervating muscles developing from the dorsal (e.g., quadriceps) and ventral muscle mass (e.g., hamstring) are situated in the lateral and medial part of the ventral gray, respectively. MN pools belonging to proximal muscles (e.g., quadratus femoris and iliopsoas) are situated ventral to those supplying more distal ones (e.g., plantar muscles). Finally, MNs innervating flexors (e.g., posterior crural muscles) are more medial than those belonging to extensors of the same joint (e.g., anterior crural muscles). These data extend and modify the MN maps in the recently published atlas of the mouse spinal cord and may help when assessing neuronal loss associated with MN diseases.

The dorsal tectal longitudinal column (TLCd): a second longitudinal column in the paramedian region of the midbrain tectum.

The tectal longitudinal column (TLC) is a longitudinally oriented, long and narrow nucleus that spans the paramedian region of the midbrain tectum of a large variety of mammals (Saldaña et al. in J Neurosci 27:13108-13116, 2007). Recent analysis of the organization of this region revealed another novel nucleus located immediately dorsal, and parallel, to the TLC. Because the name "tectal longitudinal column" also seems appropriate for this novel nucleus, we suggest the TLC described in 2007 be renamed the "ventral tectal longitudinal column (TLCv)", and the newly discovered nucleus termed the "dorsal tectal longitudinal column (TLCd)". This work represents the first characterization of the rat TLCd. A constellation of anatomical techniques was used to demonstrate that the TLCd differs from its surrounding structures (TLCv and superior colliculus) cytoarchitecturally, myeloarchitecturally, neurochemically and hodologically. The distinct expression of vesicular amino acid transporters suggests that TLCd neurons are GABAergic. The TLCd receives major projections from various areas of the cerebral cortex (secondary visual mediomedial area, and granular and dysgranular retrosplenial cortices) and from the medial pretectal nucleus. It densely innervates the ipsilateral lateral posterior and laterodorsal nuclei of the thalamus. Thus, the TLCd is connected with vision-related neural centers. The TLCd may be unique as it constitutes the only known nucleus made of GABAergic neurons dedicated to providing massive inhibition to higher order thalamic nuclei of a specific sensory modality.

Acute exercise-induced activation of Phox2b-expressing neurons of the retrotrapezoid nucleus in rats may involve the hypothalamus.

The rat retrotrapezoid nucleus (RTN) contains neurons that have a well-defined phenotype characterized by the presence of vesicular glutamate transporter 2 (VGLUT2) mRNA and a paired-like homeobox 2b (Phox2b)-immunoreactive (ir) nucleus and the absence of tyrosine hydroxylase (TH). These neurons are important to chemoreception. In the present study, we tested the hypothesis that the chemically-coded RTN neurons (ccRTN) (Phox2b(+)/TH(-)) are activated during an acute episode of running exercise. Since most RTN neurons are excited by the activation of perifornical and lateral hypothalamus (PeF/LH), a region that regulates breathing during exercise, we also tested the hypothesis that PeF/LH projections to RTN neurons contribute to their activation during acute exercise. In adult male Wistar rats that underwent an acute episode of treadmill exercise, there was a significant increase in c-Fos immunoreactive (c-Fos-ir) in PeF/LH neurons and RTN neurons that were Phox2b(+)TH(-) (p<0.05) compared to rats that did not exercise. Also the retrograde tracer Fluoro-Gold that was injected into RTN was detected in c-Fos-ir PeF/LH (p<0.05). In summary, the ccRTN neurons (Phox2b(+)TH(-)) are excited by running exercise. Thus, ccRTN neurons may contribute to both the chemical drive to breath and the feed-forward control of breathing associated with exercise.

Differential expression of VGLUT1 or VGLUT2 in the trigeminothalamic or trigeminocerebellar projection neurons in the rat.

The vesicular glutamate transporters, VGLUT1 and VGLUT2, reportedly display complementary distribution in the rat brain. However, co-expression of them in single neurons has been reported in some brain areas. We previously found co-expression of VGLUT1 and VGLUT2 mRNAs in a number of single neurons in the principal sensory trigeminal nucleus (Vp) of the adult rat; the majority of these neurons sent their axons to the thalamic regions around the posteromedial ventral nucleus (VPM) and the posterior nuclei (Po). It is well known that trigeminothalamic (T-T) projection fibers arise not only from the Vp but also from the spinal trigeminal nucleus (Vsp), and that trigeminocerebellar (T-C) projection fibers take their origins from both of the Vp and Vsp. Thus, in the present study, we examined the expression of VGLUT1 and VGLUT2 in Vp and Vsp neurons that sent their axons to the VPM/Po regions or the cortical regions of the cerebellum. For this purpose, we combined fluorescence in situ hybridization (FISH) histochemistry with retrograde tract-tracing; immunofluorescence histochemistry was also combined with anterograde tract-tracing. The results indicate that glutamatergic Vsp neurons sending their axons to the cerebellar cortical regions mainly express VGLUT1, whereas glutamatergic Vsp neurons sending their axons to the thalamic regions express VGLUT2. The present data, in combination with those of our previous study, indicate that glutamatergic Vp neurons projecting to the cerebellar cortical regions express mainly VGLUT1, whereas the majority of glutamatergic Vp neurons projecting to the thalamus co-express VGLUT1 and VGLUT2.

Co-localization of hypocretin-1 and leucine-enkephalin in hypothalamic neurons projecting to the nucleus of the solitary tract and their effect on arterial pressure.

Experiments were done to investigate whether hypothalamic hypocretin-1 (hcrt-1; orexin-A) neurons that sent axonal projections to cardiovascular responsive sites in the nucleus of the solitary tract (NTS) co-expressed leucine-enkephalin (L-Enk), and to determine the effects of co-administration of hcrt-1 and D-Ala2,D-Leu5-Enkephalin (DADL) into NTS on mean arterial pressure (MAP) and heart rate. In the first series, in the Wistar rat the retrograde tract-tracer fluorogold (FG) was microinjected (50nl) into caudal NTS sites at which L-glutamate (0.25 M; 10 nl) elicited decreases in MAP and where fibers hcrt-1 immunoreactive fibers were observed that also contained L-Enk immunoreactivity. Of the number of hypothalamic hcrt-1 immunoreactive neurons identified ipsilateral to the NTS injection site (1207 ± 78), 32.3 ± 2.3% co-expressed L-Enk immunoreactivity and of these, 2.6 ± 1.1% were retrogradely labeled with FG. Hcrt-1/L-Enk neurons projecting to NTS were found mainly within the perifornical region. In the second series, the region of caudal NTS found to contain axons that co-expressed hcrt-1 and L-Enk immunoreactivity was microinjected with a combination of hcrt-1 and DADL in α-chloralose anesthetized Wistar rats. Microinjection of DADL into NTS elicited depressor and bradycardia responses similar to those elicited by microinjection of hcrt-1. An hcrt-1 injection immediately after the DADL injection elicited an almost twofold increase in the magnitude of the depressor and bradycardia responses compared to those elicited by hcrt-1 alone. Prior injections of the non-specific opioid receptor antagonist naloxone or the specific opioid δ-receptor antagonist ICI 154,129 significantly attenuated the cardiovascular responses to the combined hcrt-1-DADL injections. Taken together, these data suggest that activation of hypothalamic-opioidergic neuronal systems contribute to the NTS hcrt-1 induced cardiovascular responses, and that this descending hypothalamo-medullary pathway may represent the anatomical substrate by which hcrt-1/L-Enk neurons function in the coordination of autonomic-cardiovascular responses during different behavioral states.