The Role of the Endogenous Opioid System in the Vocal Behavior of Songbirds and Its Possible Role in Vocal Learning.

The opioid system in the brain is responsible for processing affective states such as pain, pleasure, and reward. It consists of three main receptors, mu- (µ-ORs), delta- (δ-ORs), and kappa- (κ-ORs), and their ligands - the endogenous opioid peptides. Despite their involvement in the reward pathway, and a signaling mechanism operating in synergy with the dopaminergic system, fewer reports focus on the role of these receptors in higher cognitive processes. Whereas research on opioids is predominated by studies on their addictive properties and role in pain pathways, recent studies suggest that these receptors may be involved in learning. Rodents deficient in δ-ORs were poor at recognizing the location of novel objects in their surroundings. Furthermore, in chicken, learning to avoid beads coated with a bitter chemical from those without the coating was modulated by δ-ORs. Similarly, µ-ORs facilitate long term potentiation in hippocampal CA3 neurons in mammals, thereby having a positive impact on spatial learning. Whereas these studies have explored the role of opioid receptors on learning using reward/punishment-based paradigms, the role of these receptors in natural learning processes, such as vocal learning, are yet unexplored. In this review, we explore studies that have established the expression pattern of these receptors in different brain regions of birds, with an emphasis on songbirds which are model systems for vocal learning. We also review the role of opioid receptors in modulating the cognitive processes associated with vocalizations in birds. Finally, we discuss the role of these receptors in regulating the motivation to vocalize, and a possible role in modulating vocal learning.

The expression of DARPP-32 in adult male zebra finches (Taenopygia guttata).

Although the catecholaminergic circuitry in the zebra finch brain has been well studied, there is little information regarding the postsynaptic targets of dopamine. To answer this question, we looked at overall patterns of immunoreactivity for DARPP-32 (a dopamine and cAMP-regulated phosphoprotein, present mostly in dopaminoceptive neurons) in adult male zebra finches. Our results demonstrated that as in mammals and other avian species, DARPP-32 expression was highest in both medial and lateral striatum. Interestingly, a specific pattern of immunoreactivity was observed in the song control system, with 'core' song control regions, that is, LMANcore (lateral magnocellular nucleus of the anterior nidopallium), RA (nucleus robustus arcopallialis) and HVC being less immunoreactive for DARPP-32 than 'shell' areas such as LMANshell, RAcup, AId (intermediate arcopallium) and HVCshelf. Our results suggest that whereas dopamine may modulate the shell pathways at various levels of the AFP, dopaminergic modulation of the core pathway occurs mainly through Area X, a basal ganglia nucleus. Further, secondary sensory cortices including the perientopallial belt, Fields L1 and L3 had higher DARPP-32-immunoreactivity than primary sensory cortical areas such as the pallial basolateral nucleus, entopallium proper and Field L2, corresponding to somatosensory, visual and auditory systems, respectively. We also found DARPP-32-rich axon terminals surrounding dopaminergic neurons in the ventral tegmental area-substantia nigra complex which in turn project to the striatum, suggesting that there may be a reciprocal modulation between these regions. Overall, DARPP-32 expression appears to be higher in areas involved in integrating sensory information, which further supports the role of this protein as a molecular integrator of different signal processing pathways.

Altering Opioid Neuromodulation in the Songbird Basal Ganglia Modulates Vocalizations.

Although the interplay between endogenous opioids and dopamine (DA) in the basal ganglia (BG) is known to underlie diverse motor functions, few studies exist on their role in modulating speech and vocalization. Vocal impairment is a common symptom of Parkinson's disease (PD), wherein DA depletion affects striosomes rich in µ-opioid receptors (µ-ORs). Symptoms of opioid addiction also include deficiencies in verbal functions and speech. To understand the interplay between the opioid system and BG in vocalization, we used adult male songbirds wherein high levels of µ-ORs are expressed in Area X, a BG region which is part of a circuit similar to the mammalian thalamocortical-basal ganglia loop. Changes in DA, glutamate and GABA levels were analyzed during the infusion of different doses of the µ-OR antagonist naloxone (50 and 100 ng/ml) specifically in Area X. Blocking µ-ORs in Area X with 100 ng/ml naloxone led to increased levels of DA in this region without altering the number of songs directed toward females (FD). Interestingly, this manipulation also led to changes in the spectro-temporal properties of FD songs, suggesting that altered opioid modulation in the thalamocortical-basal ganglia circuit can affect vocalization. Our study suggests that songbirds are excellent model systems to explore how the interplay between µ-ORs and DA modulation in the BG affects speech/vocalization.

Method for improving the quality of genomic DNA obtained from minute quantities of tissue and blood samples using Chelex 100 resin.

BACKGROUND: Although genomic DNA isolation using the Chelex 100 resin is rapid and inexpensive, the DNA obtained by this method has a low concentration in solution and contains suspended impurities. The presence of debris in the DNA solution may result in degradation of DNA on long term storage and inhibition of the polymerase chain reaction. In order to remove impurities and concentrate the DNA in solution, we have introduced modifications in the existing DNA isolation protocol using Chelex-100. We used ammonium acetate to precipitate proteins and a sodium acetate- isopropanol mixture to pellet out DNA which was washed with ethanol. RESULTS: A pure DNA pellet that can be dissolved in water or Tris-EDTA buffer and stored for a long time at - 80 °C was obtained. We also observed a 20-fold change in the DNA concentration following precipitation and re-dissolution. CONCLUSION: Our method is different from other extraction methods since it uses non-toxic, easily available and inexpensive reagents as well as minimal amounts of blood or tissue samples for the DNA extraction process. Besides its use in sex determination and genotyping in lab animals as described in this paper, it may also have applications in forensic science and diagnostics such as the easy detection of pathogenic DNA in blood.

Growth and refinement of excitatory synapses in the human auditory cortex.

We had earlier demonstrated a neurofilament-rich plexus of axons in the presumptive human auditory cortex during fetal development which became adult-like during infancy. To elucidate the origin of these axons, we studied the expression of the vesicular glutamate transporters (VGLUT) 1 and 2 in the human auditory cortex at different stages of development. While VGLUT-1 expression predominates in intrinsic and cortico-cortical synapses, VGLUT-2 expression predominates in thalamocortical synapses. Levels of VGLUT-2 mRNA were higher in the auditory cortex before birth compared to postnatal development. In contrast, levels of VGLUT-1 mRNA were low before birth and increased during postnatal development to peak during childhood and then began to decrease in adolescence. Both VGLUT-1 and VGLUT-2 proteins were present in the human auditory cortex as early as 15GW. Further, immunohistochemistry revealed that the supra- and infragranular layers were more immunoreactive for VGLUT-1 compared to that in Layer IV at 34GW and this pattern was maintained until adulthood. As for VGLUT-1 mRNA, VGLUT-1 synapses increased in density between prenatal development and childhood in the human auditory cortex after which they appeared to undergo attrition or pruning. The adult pattern of VGLUT-2 immunoreactivity (a dense band of VGLUT-2-positive terminals in Layer IV) also began to appear in the presumptive Heschl's gyrus at 34GW. The density of VGLUT-2-positive puncta in Layer IV increased between prenatal development and adolescence, followed by a decrease in adulthood, suggesting that thalamic axons which innervate the human auditory cortex undergo pruning comparatively late in development.