Dopaminergic neurons develop axonal projections to their target areas in organotypic co-cultures of the ventral mesencephalon and the striatum/prefrontal cortex

Mesencephalic dopaminergic neurons are known to project to the prefrontal cortex (PFC) and the striatum (STR). Organotypic slice co-cultures of the ventral tegmental area/substantia nigra (VTA/SN)-complex and the PFC or STR, respectively, were used to analyze the cytoarchitectural organization of the VTA/SN-complex and the innervation pattern of the target slices by dopaminergic fibers. After 10-28 days of culturing immunocytochemistry with antibodies against tyrosine hydroxylase (TH) was performed. The VTA/SN-complex revealed in vitro an organization of TH-positive cells similar to those observed in rat brains of comparable age. TH-immunoreactive cells exhibited their typical morphology and formed long processes. No TH-immunolabeled elements were found in single cultures of PFC and STR. Tracing of VTA/SN fibers with biocytin as well as TH-immunostaining showed numerous labeled fibers in the co-cultured slices. Extensive fiber crossing was observed in the co-cultures of the VTA/SN-complex and STR but only a sparse fiber bridge in the co-cultured slices of VTA/SN-complex and PFC. The VTA/SN-complex-PFC system obviously retained several of its in vivo characteristics, e.g. the fiber network in the prefrontal cortical subareas. Our results demonstrate that TH-immunoreactive neurons develop their typical innervation pattern in slice co-cultures of VTA/SN-complex and PFC or STR, respectively. This in vitro approach may be useful for investigations of the dopaminergic function in the VTA/SN-prefrontal pathway.     

Dopaminergic input to GABAergic neurons in the rostral agranular insular cortex of the rat

Increasing evidence shows that the rostral agranular insular cortex (RAIC) is important in the modulation of nociception in humans and rats and that dopamine and GABA appear to be key neurotransmitters in the function of this cortical region. Here we use immunocytochemistry and path tracing to examine the relationship between dopamine and GABA related elements in the RAIC of the rat. We found that the RAIC has a high density of dopamine fibers that arise principally from the ipsilateral ventral tegmental area/substantia nigra (VTA/SN) and from a different set of neurons than those that project to the medial prefrontal cortex. Within the RAIC, there are close appositions between dopamine fibers and GABAergic interneurons. One target of cortical GABA appears to be a dense band of GABA_B receptor-bearing neurons located in lamina 5 of the RAIC. The GABA_B receptor-bearing neurons project principally to the amygdala and nucleus accumbens with few or no projections to the medial prefrontal cortex, cingulate gyrus, the mediodorsal thalamic nucleus or contralateral RAIC. The current anatomical data, together with previous behavioral results, suggest that part of the dopaminergic modulation of the RAIC occurs through GABAergic interneurons. GABA is able to exert specific effects through its action on GABA_B receptor-bearing projection neurons that target a few subcortical limbic structures. Through these connections, dopamine innervation of the RAIC is likely to affect the motivational and affective dimensions of pain.     

EphA3 null mutants do not demonstrate motor axon guidance defects

Motor axon projections are topographically ordered. Medial motor column axons project to axial muscles, whereas lateral motor column axons project to limb muscles and, along the rostrocaudal axis of the animal, the more rostral motor neuron pools project to more rostral muscle targets. We have shown that EphA3 is specifically expressed in the developing medial motor column and have postulated that EphA3 might be responsible for directing their axons to axial muscle targets. This hypothesis was supported by our demonstration that EphA3 can direct retinal ganglion cell axon targeting and by studies of ephrin-A5(-/-) mutants that show that EphA receptor signaling controls the topographic innervation of the acromiotrapezius. To test the role of EphA3 in motor axon guidance, we generated an EphA3 null mutant. Retrograde labeling studies in EphA3(-/-) embryos and adults indicate that, contrary to our predictions, EphA3 is not necessary to direct motor axons to axial muscle targets. Our results also demonstrate that ephrin A5's ability to direct topographic innervation of the acromiotrapezius must be mediated through EphA receptors other than, or in addition to, EphA3.     

Evidence for projections from medullary nuclei onto serotonergic and dopaminergic neurons in the midbrain dorsal raphe nucleus of the rat

Summary The anterograde tracer Phaseolus vulgaris-leucoagglutinin was injected into the medial nucleus of the solitary tract and into the rostral dorsomedial medulla. A sequential two-color immunoperoxidase staining was accomplished in order to demonstrate the co-distribution of presumed terminal axons with chemically distinct neurons in the dorsal raphe nucleus of the midbrain central gray, i.e., B7 serotonergic and A10dc dopaminergic neurons. Black-stained efferent fibers from the medial nucleus of the solitary tract and the rostral dorsomedial medulla intermingled with brown-stained serotonergic (5-hydroxytryptamine-immunoreactive) or dopaminergic (tyrosine hydroxylase-immunoreactive) neurons. Light microscopy revealed that the black-stained efferent axons exhibited numerous en passant and terminal varicosities that were often found in close apposition to brown-stained serotonergic and dopaminergic somata, and to proximal and distal dendrites and dendritic processes. The close association of immunoreactive elements suggests the presence of axo-somatic and axodendritic synaptic contacts of medullary fibers with serotonergic and dopaminergic neurons in the dorsal raphe nucleus. These projections could be involved in the modulation of dorsal raphe neurons, depending on the autonomic status of an animal.     

Projection of the neurons of the substantia nigra and ventral field of the midbrain tegmen in the cat to the putamen

By means of retrograde axonal transport of the horseradish peroxidase and fluorochromes in the cat, it has been stated that neurons of all the parts of the substantia nigra (SN) make projections to the putamen. These projections are organized in such a way that the rostral part of the putamen gets the projected fibers from less number of the SN parts than the caudal part. To the caudal part of this formation all parts of the SN are projected, and in the equal degree to its dorsal and ventral segments. Projections to the rostral part are sent only from two parts of the SN--compact and dorsal. To the dorsal segment of this part only axons from a small amount of the nigral neurons are sent. A small amount of neurons of the tegmental ventral field give projections to the ventral segments along the whole rostrocaudal extent of the putamen. Convergence of the SN neuronal axons in the formations of the striated body has been determined, as well as overlapping of the terminal fields in the putamen from the projective neurons of the nigral various parts. Besides in different parts of the SN discrepancy has been revealed in the neuronal populations, labelled with different stainings, that contain cells, marked with two markers, injected into the nucleus caudatus and into the putamen.     

Orexinergic Input to Dopaminergic Neurons of the Human Ventral Tegmental Area

The mesolimbic reward pathway arising from dopaminergic (DA) neurons of the ventral tegmental area (VTA) has been strongly implicated in reward processing and drug abuse. In rodents, behaviors associated with this projection are profoundly influenced by an orexinergic input from the lateral hypothalamus to the VTA. Because the existence and significance of an analogous orexigenic regulatory mechanism acting in the human VTA have been elusive, here we addressed the possibility that orexinergic neurons provide direct input to DA neurons of the human VTA. Dual-label immunohistochemistry was used and orexinergic projections to the VTA and to DA neurons of the neighboring substantia nigra (SN) were analyzed comparatively in adult male humans and rats. Orexin B-immunoreactive (IR) axons apposed to tyrosine hydroxylase (TH)-IR DA and to non-DA neurons were scarce in the VTA and SN of both species. In the VTA, 15.0±2.8% of TH-IR perikarya in humans and 3.2±0.3% in rats received orexin B-IR afferent contacts. On average, 0.24±0.05 and 0.05±0.005 orexinergic appositions per TH-IR perikaryon were detected in humans and rats, respectively. The majority (86–88%) of randomly encountered orexinergic contacts targeted the dendritic compartment of DA neurons. Finally, DA neurons of the SN also received orexinergic innervation in both species. Based on the observation of five times heavier orexinergic input to TH-IR neurons of the human, compared with the rat, VTA, we propose that orexinergic mechanism acting in the VTA may play just as important roles in reward processing and drug abuse in humans, as already established well in rodents.     

Substantia nigra dopaminergic neurons and striatal interneurons are engaged in three parallel but interdependent postnatal neurotrophic circuits

The striatum integrates motor behavior using a well‐defined microcircuit whose individual components are independently affected in several neurological diseases. The glial cell line‐derived neurotrophic factor (GDNF), synthesized by striatal interneurons, and Sonic hedgehog (Shh), produced by the dopaminergic neurons of the substantia nigra (DA SNpc), are both involved in the nigrostriatal maintenance but the reciprocal neurotrophic relationships among these neurons are only partially understood. To define the postnatal neurotrophic connections among fast‐spiking GABAergic interneurons (FS), cholinergic interneurons (ACh), and DA SNpc, we used a genetically induced mouse model of postnatal DA SNpc neurodegeneration and separately eliminated Smoothened (Smo), the obligatory transducer of Shh signaling, in striatal interneurons. We show that FS postnatal survival relies on DA SNpc and is independent of Shh signaling. On the contrary, Shh signaling but not dopaminergic striatal innervation is required to maintain ACh in the postnatal striatum. ACh are required for DA SNpc survival in a GDNF‐independent manner. These data demonstrate the existence of three parallel but interdependent neurotrophic relationships between SN and striatal interneurons, partially defined by Shh and GDNF. The definition of these new neurotrophic interactions opens the search for new molecules involved in the striatal modulatory circuit maintenance with potential therapeutic value.     

Differential responsiveness to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity in sub-regions of the primate substantia nigra and striatum

After treatment with the neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), there was a severe loss of dopamine (DA) at all examined sites in the striatum, both in those monkeys which remained asymptomatic (77-99%) and in one monkey which developed severe parkinsonian disability (94-99%). However, the asymptomatic animals had normal DA concentration in the substantia nigra (SN); yet in the symptomatic animal DA was largely depleted in the central (86%) and medial (94%), but not lateral (8%) regions of the SN. The HVA/DA ratio was raised in the striatum of all MPTP-treated animals. In the SN though, this ratio was elevated only in the symptomatic animal, in the central and medial, but not lateral regions. The contralateral half of these brains were examined for DA histofluorescence. The SN of asymptomatic animals had a slight increase in lipofuscin fluorescence within dopaminergic neurons and a small reduction in the number of dopaminergic cells, while fluorescent intensity of individual neurons was unchanged. The SN of the symptomatic animal displayed a sharp decline in the number of DA neurons along with an increase in autofluorescent pigment granules; these changes were most pronounced in the central and medial regions of the SN. These data suggest that after MPTP the terminals of the nigrostriatal pathway are affected before the cell bodies. In the one symptomatic animal emergence of parkinsonian disability corresponded with a marked loss of DA neurons and DA concentration in the central and medial regions of the SN. In the control monkeys a gradient in the concentration of amines and metabolites was observed within the SN; the lateral region contained the highest and the medial region the lowest concentration.     

Different Interactions of Prolyl Oligopeptidase and Neurotensin in Dopaminergic Function of the Rat Nigrostriatal and Mesolimbic Pathways

Prolyl oligopeptidase (PREP) is an intracellular enzyme digesting small proline-containing peptides. Since PREP resides the same brain areas as neurotensin in the nigrostriatal and mesolimbic dopaminergic pathways, we were interested to study if there is an intracellular interaction between them. A colocalization of PREP with neurotensin and neurotensin receptor 1 (NTS1) in the rat striatum, nucleus accumbens (NAcc), substantia nigra (SN) and ventral tegmental area (VTA) was studied with immunofluorescence. From the same brain areas, the levels of dopamine and its metabolites were measured 1 h after the injection of saline, NTS1 ligands (JMV-449; 5 μg) or antagonist (SR142948; 5 μg) to the rat striatum or NAcc. We also studied whether an intraperitoneal injection of a PREP inhibitor (KYP-2047; 5 mg/kg) affects the levels of dopamine and its metabolites alone or modifies the effects of the NTS1 ligands. PREP was highly colocalized with neurotensin and NTS1 in the VTA, and with NTS1 in the SN. Colocalization was moderate or low in other brain areas. When injected to the striatum, JMV-449 had a tendency to increase dopamine (p = 0.052) and metabolite levels in the striatum and SN, whereas SR142948 did not. After the injection to the NAcc, JMV-449 but not SR142948, increased dopamine levels in the VTA and dopamine metabolite levels in the NAcc and VTA. KYP-2047 decreased the dopamine levels in the striatum, but increased dopamine metabolite levels in the NAcc and VTA. Our results suggest a novel role for PREP in the modulation of dopaminergic transmission, which may be different in nigrostriatal and mesolimbic pathways.     

Different profiles of main and accessory olfactory bulb mitral/tufted cell projections revealed in mice using an anterograde tracer and a whole-mount, flattened cortex preparation

A whole-mount, flattened cortex preparation was developed to compare profiles of axonal projections from main olfactory bulb (MOB) and accessory olfactory bulb (AOB) mitral and tufted (M/T) cells. After injections of the anterograde tracer, Phaseolus vulgaris leucoagglutinin, mapping of labeled axons using a Neurolucida system showed that M/T cells in the AOB sent axons primarily to the medial and posterior lateral cortical amygdala, with minimal branching into the piriform cortex. By contrast, M/T cells in the MOB displayed a network of collaterals that branched off the primary axon at several levels of the lateral olfactory tract (LOT). Collaterals emerging from the LOT into the anterior piriform cortex were often observed crossing into the posterior piriform cortex. M/T cells in the dorsal MOB extended fewer collaterals from the primary axon in the rostral LOT than did M/T cells from the anterior or ventral MOB. MOB M/T cells that projected to the medial amygdala did not do so exclusively, also sending collaterals to the anterior cortical amygdala as well as to olfactory cortical regions. This arrangement may be related to the ability of social experience to modify the response of mice to volatile pheromones detected by the main olfactory system.     

Dissection and Culture of Mouse Dopaminergic and Striatal Explants in Three-Dimensional Collagen Matrix Assays

Midbrain dopamine (mdDA) neurons project via the medial forebrain bundle towards several areas in the telencephalon, including the striatum¹. Reciprocally, medium spiny neurons in the striatum that give rise to the striatonigral (direct) pathway innervate the substantia nigra². The development of these axon tracts is dependent upon the combinatorial actions of a plethora of axon growth and guidance cues including molecules that are released by neurites or by (intermediate) target regions^3,4. These soluble factors can be studied in vitro by culturing mdDA and/or striatal explants in a collagen matrix which provides a three-dimensional substrate for the axons mimicking the extracellular environment. In addition, the collagen matrix allows for the formation of relatively stable gradients of proteins released by other explants or cells placed in the vicinity (e.g. see references 5 and 6). Here we describe methods for the purification of rat tail collagen, microdissection of dopaminergic and striatal explants, their culture in collagen gels and subsequent immunohistochemical and quantitative analysis. First, the brains of E14.5 mouse embryos are isolated and dopaminergic and striatal explants are microdissected. These explants are then (co)cultured in collagen gels on coverslips for 48 to 72 hours in vitro. Subsequently, axonal projections are visualized using neuronal markers (e.g. tyrosine hydroxylase, DARPP32, or βIII tubulin) and axon growth and attractive or repulsive axon responses are quantified. This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of mesostriatal and striatonigral axon growth and guidance during development. Using this assay, it is also possible to assess other (intermediate) targets for dopaminergic and striatal axons or to test specific molecular cues.     

Individual differences in the motivation to communicate relate to levels of midbrain and striatal catecholamine markers in male European starlings

Individuals display dramatic differences in social communication even within similar social contexts. Across vertebrates dopaminergic projections from the ventral tegmental area (VTA) and midbrain central gray (GCt) strongly influence motivated, reward-directed behaviors. Norepinephrine is also rich in these areas and may alter dopamine neuronal activity. The present study was designed to provide insight into the roles of dopamine and norepinephrine in VTA and GCt and their efferent striatal target, song control region area X, in the regulation of individual differences in the motivation to sing. We used high pressure liquid chromatography with electrochemical detection to measure dopamine, norepinephrine and their metabolites in micropunched samples from VTA, GCt, and area X in male European starlings (Sturnus vulgaris). We categorized males as sexually motivated or non-sexually motivated based on individual differences in song produced in response to a female. Dopamine markers and norepinephrine in VTA and dopamine in area X correlated positively with sexually-motivated song. Norepinephrine in area X correlated negatively with non-sexually-motivated song. Dopamine in GCt correlated negatively with sexually-motivated song, and the metabolite DOPAC correlated positively with non-sexually-motivated song. Results highlight a role for evolutionarily conserved dopaminergic projections from VTA to striatum in the motivation to communicate and highlight novel patterns of catecholamine activity in area X, VTA, and GCt associated with individual differences in sexually-motivated and non-sexually-motivated communication. Correlations between dopamine and norepinephrine markers also suggest that norepinephrine may contribute to individual differences in communication by modifying dopamine neuronal activity in VTA and GCt.     

The hTH-GFP Reporter Rat Model for the Study of Parkinson's Disease

Parkinson disease (PD) is the second leading neurodegenerative disease in the US. As there is no known cause or cure for PD, researchers continue to investigate disease mechanisms and potential new therapies in cell culture and in animal models of PD. In PD, one of the most profoundly affected neuronal populations is the tyrosine hydroxylase (TH)-expressing dopaminergic (DA) neurons of the substantia nigra pars compacta (SNpc). These DA-producing neurons undergo degeneration while neighboring DA-producing cells of the ventral tegmental area (VTA) are largely spared. To aid in these studies, The Michael J. Fox Foundation (MJFF) partnered with Thomas Jefferson University and Taconic Inc. to generate new transgenic rat lines carrying the human TH gene promoter driving EGFP using a 11 kb construct used previously to create a hTH-GFP mouse reporter line. Of the five rat founder lines that were generated, three exhibited high level specific GFP fluorescence in DA brain structures (ie. SN, VTA, striatum, olfactory bulb, hypothalamus). As with the hTH-GFP mouse, none of the rat lines exhibit reporter expression in adrenergic structures like the adrenal gland. Line 12141, with its high levels of GFP in adult DA brain structures and minimal ectopic GFP expression in non-DA structures, was characterized in detail. We show here that this line allows for anatomical visualization and microdissection of the rat midbrain into SNpc and/or VTA, enabling detailed analysis of midbrain DA neurons and axonal projections after toxin treatment in vivo. Moreover, we further show that embryonic SNpc and/or VTA neurons, enriched by microdissection or FACS, can be used in culture or transplant studies of PD. Thus, the hTH-GFP reporter rat should be a valuable tool for Parkinson''s disease research.     

Sonic hedgehog is indirectly required for intraretinal axon pathfinding by regulating chemokine expression in the optic stalk

Successful axon pathfinding requires both correct patterning of tissues, which will later harbor axonal tracts, and precise localization of axon guidance cues along these tracts at the time of axon outgrowth. Retinal ganglion cell (RGC) axons grow towards the optic disc in the central retina, where they turn to exit the eye through the optic nerve. Normal patterning of the optic disc and stalk and the expression of guidance cues at this choice point are necessary for the exit of RGC axons out of the eye. Sonic hedgehog (Shh) has been implicated in both patterning of ocular tissue and direct guidance of RGC axons. Here, we examine the precise spatial and temporal requirement for Hedgehog (Hh) signaling for intraretinal axon pathfinding and show that Shh acts to pattern the optic stalk in zebrafish but does not guide RGC axons inside the eye directly. We further reveal an interaction between the Hh and chemokine pathways for axon guidance and show that cxcl12a functions downstream of Shh and depends on Shh for its expression at the optic disc. Together, our results support a model in which Shh acts in RGC axon pathfinding indirectly by regulating axon guidance cues at the optic disc through patterning of the optic stalk.     

Motoneuron projection patterns in chick embryonic limbs with a double complement of dorsal thigh musculature

During the normal development of the chick, lateral motoneurons within the lumbosacral motor column of the spinal cord consistently project to muscles of dorsal origin within the limb while medial motoneurons project to muscles of ventral origin. To determine if specific cues arising from each type of target are the dominant guidance cues used by lateral and medial motoneurons to create this pattern, I examined motoneuron projections in embryonic chick limbs with a double complement of dorsal thigh musculature and no ventral musculature. Results indicate that cues associated with muscles of a specific developmental origin do not invariably dominate. Before and after the major period of motoneuron death, all muscles in dorsal limb regions (host) were innervated by lateral or dorsal pool neurons. Most ventrally positioned (donor) muscles were innervated by medial or ventral pool neurons. Only the donor iliofibularis, a muscle located very near to its original source of innervation, received projections from some lateral neurons. Within the limb proper, medial or ventral pool neurons projected to donor muscles in a patterned manner suggesting that they were following nonspecific regional cues and perhaps also responding to the availability of uninnervated target tissue. I conclude that axon sorting into distinct lateral and medial classes is independent of limb target complement and that subsequent pathway choice is a separate event governed by both specific target cues and other guidance mechanisms.     

Unique properties of mesoprefrontal neurons within a dual mesocorticolimbic dopamine system

The mesocorticolimbic dopamine system is essential for cognitive and emotive brain functions and is thus an important target in major brain diseases like schizophrenia, drug addiction, and attention deficit hyperactivity disorder. However, the cellular basis for the diversity in behavioral functions and associated dopamine-release pattern within the mesocorticolimbic system has remained unclear. Here, we report the identification of a type of dopaminergic neuron within the mesocorticolimbic dopamine system with unconventional fast-firing properties and small DAT/TH mRNA expression ratios that selectively projects to prefrontal cortex and nucleus accumbens core and medial shell as well as to basolateral amygdala. In contrast, well-described conventional slow-firing dopamine midbrain neurons only project to the lateral shell of the nucleus accumbens and the dorsolateral striatum. Among this dual dopamine midbrain system defined in this study by converging anatomical, electrophysiological, and molecular properties, mesoprefrontal dopaminergic neurons are unique, as only they do not possess functional somatodendritic Girk2-coupled dopamine D2 autoreceptors.     

Intrastriatal administration of human immunodeficiency virus-1 glycoprotein 120 reduces glial cell-line derived neurotrophic factor levels and causes apoptosis in the substantia nigra

Uninfected neurons of the substantia nigra (SN) degenerate in human immunodeficiency virus (HIV)-positive patients through an unknown etiology. The HIV envelope glycoprotein 120 (gp120) causes apoptotic neuronal cell death in the rodent striatum, but its primary neurotoxic mechanism is still under investigation. Previous studies have shown that gp120 causes neurotoxicity in the rat striatum by reducing brain-derived neurotrophic factor (BDNF). Because glial cell line-derived neurotrophic factor (GDNF) and BDNF are neurotrophic factors crucial for the survival of dopaminergic neurons of the SN, we investigated whether gp120 reduces GDNF and BDNF levels concomitantly to induce apoptosis. Rats received a microinjection of gp120 or vehicle into the striatum and were sacrificed at various time intervals. GDNF but not BDNF immunoreactivity was decreased in the SN by 4 days in gp120-treated rats. In these animals, a significant increase in the number of caspase-3- positive neurons, both tyrosine hydroxylase (TH)-positive and -negative, was observed. Analysis of TH immunoreactivity revealed fewer TH-positive neurons and fibers in a medial and lateral portion of cell group A9 of the SN, an area that projects to the striatum, suggesting that gp120 induces retrograde degeneration of nigrostriatal neurons. We propose that dysfunction of the nigrostriatal dopaminergic system associated with HIV may be caused by a reduction of neurotrophic factor expression by gp120.     

Ephrin‐A5 regulates the formation of the ascending midbrain dopaminergic pathways

Dopaminergic neurons from the substantia nigra and the ventral tegmental area of the midbrain project to the caudate/putamen and nucleus accumbens, respectively, establishing the mesostriatal and the mesolimbic pathways. However, the mechanisms underlying the development of these pathways are not well understood. In the current study, the EphA5 receptor and its corresponding ligand, ephrin‐A5, were shown to regulate dopaminergic axon outgrowth and influence the formation of the midbrain dopaminergic pathways. Using a strain of mutant mice in which the EphA5 cytoplasmic domain was replaced with β‐galactosidase, EphA5 protein expression was detected in both the ventral tegmental area and the substantia nigra of the midbrain. Ephrin‐A5 was found in both the dorsolateral and the ventromedial regions of the striatum, suggesting a role in mediating dopaminergic axon‐target interactions. In the presence of ephrin‐A5, dopaminergic neurons extended longer neurites in in vitro coculture assays. Furthermore, in mice lacking ephrin‐A5, retrograde tracing studies revealed that fewer neurons sent axons to the striatum. These observations indicate that the interactions between ephrin‐A ligands and EphA receptors promote growth and targeting of the midbrain dopaminergic axons to the striatum. © 2008 Wiley Periodicals, Inc. Develop Neurobiol, 2009