Eszter Bálint

Efferent connections of nucleus accumbens subdivisions of the domestic chicken (Gallus domesticus): An anterograde pathway tracing study

Envisaged as a limbic‐motor interface, the mammalian nucleus accumbens (Ac) is responsible for motivation, emotionality, and reward mechanisms. As in mammals, Ac of the domestic chick has three subdivisions: the rostral pole (AcR) lying in the rostral part of basal telencephalon, the core (AcC), corresponding to the ventromedial medial striatum, and the shell (AcS), lying ventrally and ventrolaterally to the AcC. Less well known is the connectivity of subdivisions. Here we report on the efferents of Ac subregions, using biotinylated dextran amine as anterograde tracer, deposited into the AcR, AcS, and AcC. The projections of the accumbens subregions mainly overlap in the telencephalon and the diencephalon but differ in the brainstem. In the telencephalon, the main projection sites are the ventral pallidum, the basal nucleus (Meynert), and the nucleus of the diagonal band. The lateral hypothalamus and lateral preoptic area receive strong projections from the AcR and AcS, and weaker projections from the AcC. The AcR and AcC massively innervate the subthalamic nucleus. In the brainstem the bulk of accumbens fibers were found in the compact part of the substantia nigra. All subregions project to the parabrachial region, reticular formation, periaqueductal gray, and the raphe nuclei, with some differences in the weights and subregional distributions. AcR and AcS project extensively to the ventral tegmental area, while AcC sends massive innervation to the solitary and vagal motor nuclei. Overall, the results seem to support the previously suggested distribution of Ac subregions, emphasizing similarities and differences with mammals. J. Comp. Neurol. 519:2922–2953, 2011.

Afferent connections of septal nuclei of the domestic chick (Gallus domesticus): A retrograde pathway tracing study

The afferents to the septum of the domestic chicken were studied using retrograde tracers, rhodamine conjugated latex bead or Fast Blue, placed in different septal subregions. The results were verified by anterograde tracer injections deposited to selected areas. The main telencephalic afferents to the septum arise ipsilaterally from the hippocampal formation, dorsolateral corticoid area, piriform cortex, amygdaloid pallium, and the ventral pallidum. Contralateral afferents originate from the lateral septum and the amygdaloid pallium. A massive bilateral projection arises from the lateral hypothalamus. Other hypothalamic afferents arise from the periventricular, paraventricular and anterior medial nuclei, and the premammillary and mammillary areas. The dorsal thalamic nuclei (dorsal medial anterior and posterior) and the reticular dorsal nuclei also contribute septal afferents. Brainstem afferents arise bilaterally from the ventral tegmental area, substantia nigra, central gray, A8, locus coeruleus, ventral subcoeruleus nucleus, and raphe nuclei. The main terminal fields for septal afferents lie in the lateral septal nucleus and the belt of medial septal nucleus. The core of the latter is invaded mainly by fibers from the brainstem, presumably belonging to the ascending activating system. The septal afferents of the chicken are largely similar to those of other avian and nonavian species. The most prominent differences with previous pigeon data were found in the subregional selectivity of the hippocampal formation, dorsolateral corticoid area, mammillary nuclei, some dorsal thalamic nuclei, substantia nigra, and subcoeruleus nuclei in their projections to defined septal nuclei. J. Comp. Neurol. 511:109–150, 2008.

Postnatal changes in the distribution and density of neuronal nuclei and doublecortin antigens in domestic chicks (Gallus domesticus)

To understand better the rate of neurogenesis and the distribution of new neurons in posthatch domestic chicks, we describe and compare the expression of the neuronal nuclei protein (NeuN, a.k.a. Fox‐3) and doublecortin antigens in the whole brain of chicks 2 days, 8 days, and 14 weeks posthatch. In the forebrain ventricular and paraventricular zones, the density of bromodeoxyuridine‐, NeuN‐, and doublecortin‐labeled cells was compared between chicks 24 hours and 7 days after an injection of bromodeoxyuridine (2 and 8 days posthatch, respectively). The distribution of NeuN‐labeled neurons was similar to Nissl‐stained tissue, with the exception of some areas where neurons did not express NeuN: cerebellar Purkinje cells and olfactory bulb mitral cells. The ventral tegmental area of 2‐day‐old chicks was also faintly labeled. The distribution of doublecortin was similar at all timepoints, with doublecortin‐labeled profiles located throughout all forebrain areas as well as in the cerebellar granule cell layer. However, doublecortin labeling was not detectable in any midbrain or brainstem areas. Our data indicate that a significant number of new neurons is still formed in the telencephalon of posthatch domestic chicks, whereas subtelencephalic areas (except for the cerebellum) finish their neuronal expansion before hatching. Most newly formed cells in chicks leave the paraventricular zone after hatching, but a pool of neurons stays in the vicinity of the ventricular zone and matures in situ within 7 days. Proliferating cells often migrate laterally along forebrain laminae into still‐developing brain areas. J. Comp. Neurol., 2012.

The organisation of the basal ganglia in the domestic chick (Gallus domesticus): Anatomical localisation of DARPP-32 in relation to glutamate

Subpallial structures are highly conserved across the different vertebrate species. They are instrumental in the neural processing relevant to adaptive learning, decision making, motivation and behavioural strategies. Of the striatal regions, our attention has been focussed on the medial and ventral striatum (MSt), now parcellated into subregions, and also including the nucleus accumbens (Ac). Similar to mammals, the avian Ac and MSt receive glutamatergic input from the pallium and dopaminergic input from the substantia nigra and ventral tegmental area. Coincidence between glutamatergic and dopaminergic synaptic activities in the ventral/medial striatum, including the Ac, is required for memory to be formed for a given pairing of stimulus and a hedonic quality or behavioural salience. The underlying mechanism involves the activation of NMDA and dopaminergic receptors, as well as the phosphorylation of dopamine-cAMP-regulated phosphoprotein (DARPP-32). Using quantitative electron microscopy of chick specimens double-labelled against glutamate and DARPP-32 we observed direct synaptic connections between glutamate immunoreactive axon terminals and DARPP-32 labelled dendrites in the MSt and also in the posterolateral telencephalon (nidopallium caudolaterale, a prefrontal cortex equivalent region) and the hippocampus. Glutamate immunoreactive axons synapsed with both DARPP-32 immunoreactive (DARPP-32+) and DARPP-32 negative (DARPP-32-) dendrites, forming asymmetrical junctions, in all brain regions observed. The existence of direct synaptic contacts between excitatory amino acid containing axon terminals and DARPP-32 containing dopaminoceptive neurons of the chicken MSt underlines the functional homology with mammalian striatal systems.

Abundance and location of DARPP-32 in striato-tegmental circuits of domestic chicks.

The striatum is reciprocally connected to the brainstem dopaminergic nuclei and receives a strong dopaminergic input. In the present study the spatial relation between the dopaminergic and dopaminoceptive structures of the avian medial striatum (formerly: lobus parolfactorius) was observed by confocal laser scanning microscope in the domestic chick (Gallus domesticus). We also analysed the connections in the area ventralis tegmentalis and the substantia nigra. To label the dopaminergic structures, anti-tyrosine hydroxylase was used and DARPP-32 (dopamine and cAMP regulated phosphoprotein) was a marker of dopaminoceptive elements. The tyrosine hydroxylase positive fibres formed baskets of juxtapositions around the DARPP-32 containing cells of the medial striatum. However, such baskets were also observed to juxtapose DARPP-32 immunonegative cells. In the tegmentum, DARPP-32 was observed in axons descending from the telencephalon via the ansa lenticularis. These varicose fibers innervated the ventral tegmental area and substantia nigra and were often juxtaposed to dopaminergic neurons and dendrites. Approximately 40% of the striatal projection neurons targeting the ventral tegmentum, and 60% of striatal projection neurons targeting the nigra were immunoreactive to DARPP-32, as revealed by retrograde pathway tracing with Fast Blue. Endogenous dopamine may exert a retrograde synaptic effect on the afferent striato-tegmental fibers, apart from the reported extrasynaptic action. The abundance of juxtapositions observed in the avian brainstem and medial striatum corroborates the possibility of reciprocal striato-tegmental circuits, relevant to the reinforcement of behaviour.

Amygdalofugal axon terminals immunoreactive for L-aspartate or L-glutamate in the nucleus accumbens of rats and domestic chickens: A comparative electron microscopic immunocytochemical study combined with anterograde pathway tracing

Several studies have shown that L-aspartate (Asp) is present in synaptic vesicles and released exocytotically from presynaptic terminals, possibly by Ca2+-dependent corelease of Asp and L-glutamate (Glu). It has been demonstrated that both excitatory amino acids (EAAs) are released from the rat striatum as part of corticostriatal neurotransmission. The single or colocalized occurrence of Asp and Glu in specific synaptic boutons of the chicken medial striatum/nucl. accumbens has been demonstrated by our group using ultrastructural immunocytochemistry. However, evidence for the presence of EAAs in any specific striatal pathway was only circumstantial. Here, we report on the distribution of Asp and Glu in specific synaptic terminals of the amygdalostriatal pathway, both in rat and chicken brains, combining anterograde tracing with postembedding immunogold labeling of Asp or Glu. Immunoreactivity for Asp and Glu was observed in amygdalofugal terminals with asymmetrical synaptic junctions (morphologically representing excitatory synapses) in both species. The postsynaptic targets were either dendritic spines or small dendrites, whereas axosomatic or axo-axonic connections were not observed. Ultrastructurally, the synaptic terminals immunoreactive for Asp were indistinguishable from those immunoreactive for Glu. The findigs are consistent with an Asp–Glu corelease mechanism, with a distinct synaptic contingent, evolutionarily conserved in the amygdalostriatal pathway.

Neurotensin: revealing a novel neuromodulator circuit in the nucleus accumbens–parabrachial nucleus projection of the domestic chick

Lower brainstem projections from nucleus accumbens (Ac) subregions to the parabrachial complex (PB), the nucleus of the solitary tract and the vagal motor nuclei have been described previously in the domestic chick by our group. Such projections, particulary those from the core and rostral pole regions of Ac have not been found in mammals or pigeons. Here we report on the presence of neurotensin (NT) in the neurons projecting from different Ac subnuclei, and also from the bed nucleus of stria terminalis, to the PB in the domestic chicken. The study is based upon correlated retrograde tracing (using Fast Blue) and NT immunohistochemistry, supplemented with regional charting and quantitative analysis of double-labeled neurons. The number of retrogradely labeled cells in Ac subdivisions reflects the size of FB tracer deposit, and the degree to which it extends to the medial PB. Of all Ac subregions, the core contained the largest amount of double-labeled cells. The findings demonstrate that the anatomical pathway through which the Ac can directly modulate taste-responsive neurons of the PB employs mainly neurotensin as a neuromodulator. The observed anatomical difference between mammals and birds is either a general taxonomic feature or it reflects feeding strategies specific for the domestic chick. The results are also relevant to a better understanding of the role of NT in food intake and reward-related behaviors in birds.

Age-related and function-dependent regional alterations of free L- and D-aspartate in postembryonic chick brain

D-aspartate (D-Asp) modulates adult neural plasticity and embryonic brain development by promoting cell proliferation, survival and differentiation. Here, developmental changes of the excitatory amino acids (EAAs) L-Glu, L-Asp and D-Asp were determined during the first postembryonic days, a time window for early learning, in selected brain regions of domestic chickens after chiral separation and capillary electrophoresis. Extracellular concentration (ECC) of EAAs was measured in microdialysis samples from freely moving chicks. ECC of D-Asp (but not L-EAAs) decreased during the first week of age, with no considerable regional or learning-related variation. ECC of L-Asp and L-Glu (but not of D-Asp) were elevated in the mSt/Ac in response to a rewarding stimulus, suggesting importance of Asp-Glu co-release in synaptic plasticity of basal ganglia. Potassium-evoked release of D-Asp, with a protracted transient, was also demonstrated. D-Asp constitutes greater percentage of total aspartate in the extracellular space than in whole tissue extracts, thus the bulk of D-Asp detected in tissue appears in the extracellular space. Conversely, only a fraction of tissue L-EAAs can be detected in extracellular space. The lack of changes in tissue D-Asp following avoidance learning indicates a tonic, rather than phasic, mechanism in the neuromodulatory action of this amino acid.