Combined Retrograde Labeling Research Articles

Histochemical Characterization of the Dorsal Raphe-Periaqueductal Grey Dopamine Transporter Neurons Projecting to the Extended Amygdala.

The dorsal raphe (DR) nucleus contains many tyrosine hydroxylase (TH)-positive neurons which are regarded as dopaminergic (DA) neurons. These DA neurons in the DR and periaqueductal gray (PAG) region (DADR-PAG neurons) are a subgroup of the A10 cluster, which is known to be heterogeneous. This DA population projects to the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST) and has been reported to modulate various affective behaviors. To characterize, the histochemical features of DADR-PAG neurons projecting to the CeA and BNST in mice, the current study combined retrograde labeling with Fluoro-Gold (FG) and histological techniques, focusing on TH, dopamine transporter (DAT), vasoactive intestinal peptide (VIP), and vesicular glutamate transporter 2 (VGlut2). To identify putative DA neurons, DAT-Cre::Ai14 mice were used. It was observed that DATDR-PAG neurons consisted of the following two subpopulations: TH+/VIP– and TH–/VIP+ neurons. The DAT+/TH–/VIP+ subpopulation would be non-DA noncanonical DAT neurons. Anterograde labeling of DATDR-PAG neurons with AAV in DAT-Cre mice revealed that the fibers exclusively innervated the lateral part of the CeA and the oval nucleus of the BNST. Retrograde labeling with FG injections into the CeA or BNST revealed that the two subpopulations similarly innervated these regions. Furthermore, using VGlut2-Cre::Ai14 mice, it was turned out that the TH–/VIP+ subpopulations innervating both CeA and BNST were VGlut2-positive neurons. These two subpopulations of DATDR-PAG neurons, TH+/VIP– and TH–/VIP+, might differentially interfere with the extended amygdala, thereby modulating affective behaviors.

Chronic intermittent ethanol exposure dysregulates a GABAergic microcircuit in the bed nucleus of the stria terminalis

Neuroadaptations in brain regions that regulate emotional and reward-seeking behaviors have been suggested to contribute to pathological behaviors associated with alcohol-use disorder. One such region is the bed nucleus of the stria terminalis (BNST), which has been linked to both alcohol consumption and alcohol withdrawal-induced anxiety and depression. Recently, we identified a GABAergic microcircuit in the BNST that regulates anxiety-like behavior. In the present study, we examined how chronic alcohol exposure alters this BNST GABAergic microcircuit in mice. We selectively targeted neurons expressing corticotropin releasing factor (CRF) using a CRF-reporter mouse line and combined retrograde labeling to identify BNST projections to the ventral tegmental area (VTA) and lateral hypothalamus (LH). Following 72 h of withdrawal from four weekly cycles of chronic intermittent ethanol (CIE) vapor exposure, the excitability of a sub-population of putative local CRF neurons that did not project to either VTA or LH (CRFnon−VTA/LH neurons) was increased. Withdrawal from CIE also increased excitability of non-CRF BNST neurons that project to both LH and VTA (BNSTnon−CRF-proj neurons). Furthermore, both populations of neurons had a reduction in spontaneous EPSC amplitude while frequency was unaltered. Withdrawal from chronic alcohol was accompanied by a significant increase in spontaneous IPSC frequency selectively in the BNSTnon−CRF-proj neurons. Together, these data suggest that withdrawal from chronic ethanol dysregulates local CRF-GABAergic microcircuit to inhibit anxiolytic outputs of the BNST which may contribute to enhanced anxiety during alcohol withdrawal and drive alcohol-seeking behavior.This article is part of the special issue on ‘Neuropeptides’.

Correlated Gene Expression and Target Specificity Demonstrate Excitatory Projection Neuron Diversity

The neocortex contains diverse populations of excitatory neurons segregated by layer and further definable by their specific cortical and subcortical projection targets. The current study describes a systematic approach to identify molecular correlates of specific projection neuron classes in mouse primary somatosensory cortex (S1), using a combination of in situ hybridization (ISH) data mining, marker gene colocalization, and combined retrograde labeling with ISH for layer-specific marker genes. First, we identified a large set of genes with specificity for each cortical layer, and that display heterogeneous patterns within those layers. Using these genes as markers, we find extensive evidence for the covariation of gene expression and projection target specificity in layer 2/3, 5, and 6, with individual genes labeling neurons projecting to specific subsets of target structures. The combination of gene expression and target specificity imply a great diversity of projection neuron classes that is similar to or greater than that of GABAergic interneurons. The covariance of these 2 phenotypic modalities suggests that these classes are both discrete and genetically specified.

Demonstration of a trigeminothalamic pathway to the oval paracentral intralaminar thalamic nucleus and its involvement in the processing of noxious orofacial deep inputs

Using combined retrograde labeling and Fos protein immunohistochemistry, we show that after masseter inflammation, a population of neurons in the dorsal portion of the subnuclei interpolaris/caudalis transition zone at the level of the obex was activated and projected to the oval paracentral nucleus (OPC) of the intralaminar thalamic nuclei. The present findings indicate a trigeminothalamic pathway to the OPC intralaminar nucleus involved in central processing of orofacial deep noxious input.

Serotonergic pathways in the CNS of Aplysia californica: analysis of projections onto the pedal ganglia by combined retrograde labelling & immunocytochemistry

Serotonergic pathways in the CNS of Aplysia californica: analysis of projections onto the pedal ganglia by combined retrograde labelling & immunocytochemistry

A catecholaminergic projection from the ventral tegmental area to the diagonal band of broca: Modulation by neurotensin

The ventral tegmental area contains a high density of dopaminergic perikarya having ascending projections to a number of limbic forebrain regions. In this study, we use combined retrograde labeling with horseradish peroxidase (HRP) and immunohistochemical staining for tyrosine hydroxylase to examine the catecholaminergic projection from the ventral tegmental area to the diagonal band of Broca. When injection of HRP was restricted to the diagonal band, only neurons in the nucleus linearis, nucleus interfascicularis and ventromedial portion of the nucleus paranigralis were labeled. In contrast, HRP injection into the adjacent nucleus accumbens labeled neurons throughout these nuclei, plus the nucleus parabrachialis pigmentosus, nucleus retroruber and substantia nigra, pars compacta. Approximately 60% of neurons in the ventral tegmental area labeled from the diagonal band contained tyrosine hydroxylase, compared with 79% of the neurons labeled from the nucleus accumbens. Neurotensin is a tridecapeptide found in the ventral tegmental area which has been shown to activate dopamine neurons projecting to the nucleus accumbens. In this study, microinjection of neurotensin into ventral tegmental nuclei which contained neurons retrogradely labeled from the diagonal band significantly elevated the levels of dopamine metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, in the diagonal band. The results of this study demonstrate that a catecholaminergic projection exists from the ventral tegmental area to the diagonal band of Broca, and that this pathway can be stimulated by intra-ventral tegmental injection with neurotensin.

The projections of the ventral tegmental area and adjacent regions: A combined fluorescent retrograde tracer and immunofluorescence study in the rat

The organization of projection neurons in the ventral tegmental area (VTA), and in adjacent parts of the raphe nuclei medial to it (the central and rostral linear, and interfascicular nuclei), the mammillary body (the supramammillary region and the tuberomammillary nucleus), and the substantia nigra have been examined in the rat with Kuypers' retrograde double labeling method, and with a combined retrograde labeling (with true blue)-immunohistochemical method for the demonstration of dopaminergic neurons. First, the distribution, within the VTA and adjacent regions, of dopaminergic and non-dopaminergic cells that project to terminal fields in the telencephalon (nucleus accumbens, lateral septum, pre- and supragenual fields of the anterior limbic cortex, amygdala, dorsal hippocampus, and entorhinal area), in the diencephalon (lateral habenula), and in the brainstem (locus coeruleus, and parabrachial nucleus) was determined. Then, 15 different combinations of injections of the tracers bisbenzimide and true blue into different terminal fields were made to determine whether individual cells in the region of the VTA send collaterals to more than one site. Taken together, the results indicate that essentially separate groups of cells in the VTA and adjacent regions of the raphe project to each terminal field. In addition, each group can be further divided into dopaminergic and non-dopaminergic components, although the proportion of dopaminergic cells in each group can vary from over 80% (e.g., to the nucleus accumbens) to less than 1% (to the lateral habenula and to the locus coeruleus). In addition, it was found that the supramammillary region, which contains a dense extension of the A10 cell group in its medial part, and the tuberomammillary nucleus, project to, or through, most of the regions injected with retrograde tracers. Virtually all of the projections from the VTA and adjacent regions are partially crossed, the percentage of cells on the uninjected side ranging from over 40% (e.g., for locus coeruleus injections) to only about 2% (e.g., for amygdalar injections). Most of the groups of projection neurons in the region of the VTA are considerably intermixed with the exception of those that project to the lateral septum, to the lateral habenula, and to the hippocampal formation, which are concentrated in ventral and medial parts of the VTA, and in the raphe nuclei medial to the VTA. It was concluded that in the ventral part of the midbrain, essentially separate groups of aminergic and non-aminergic neurons in both the reticular formation (VTA) and in the adjacent nuclei of the raphe project bilaterally to a variety of similar terminal fields in the telencephalon, diencephalon, and brainstem. Further work at the single cell level is needed to determine whether these cell groups are differentially innervated by known inputs to the VTA and adjacent regions, most of which appear to descend through the medial forebrain bundle from sites in the limbic system and hypothalamus.