song is a complex , learned motor skill that involves the precise coordination of vocal and respiratory musculature in order to produce highly stereotyped renditions of a memorized song model .
two pathways contributed to this behavior are identified in the songbird forebrain : the motor pathway that is required throughout life for normal song production , and the anterior forebrain pathway ( afp ) which is necessary for song learning and plasticity [ 13 ] .
hvc ( used as the proper name ) is the beginning nucleus of both pathways and projects to the robust nucleus of the arcopallium ( ra ) and afp .
the motor pathway is from hvc to ra , and then ra projects to brainstem nuclei controlling the vocal and respiratory muscles . during song , ra neurons in adult birds generate a highly stereotyped sequence of bursts [ 5 , 6 ] and also receive input from lateral magnocellular nucleus of the anterior nidopallium ( lman ) , the output nucleus of afp .
notably , recordings from lman neurons projecting to ra revealed highly variable spiking activity across song renditions , suggesting that lman may act as a source of variability .
these results indicate that synaptic activity in lman - ra synaptic transmission is crucial to song plasticity [ 8 , 9 ] , but the detailed mechanism is unclear . it has been observed that ra received excitatory glutamatergic inputs from hvc and lman .
the former is mediated by a mixture of n - methyl - d - aspartate ( nmda ) and -amino-3-hydroxy-5-methyl-4-isoxazolepropionic - acid- ( ampa- ) type receptors [ 4 , 10 , 11 ] , and the latter is mediated almost exclusively by nmda - type receptors .
lman - ra synaptic transmission is necessary for the variability of song in adult zebra finch , suggesting that the activity of nmda receptor on the lman - ra synapses might induce the activity pattern change of ra neurons and change the bursts that control the vocal and respiratory muscles .
the control of gain , which refers to modulation of a neuron 's responsiveness to input , is critically important for normal sensory , cognitive , and motor functions [ 1215 ] .
gain control is achieved through a divisive process and is observed as a change in the slope of the input - output relationship .
such gain changes are frequently observed in the responses of cortical neurons and are thought to play an important role in neural computations .
modulations of gain have been observed in the enhancement of neural responses by attention [ 12 , 16 ] and can also be induced pharmacologically . in a neuron model ,
nmda receptors caused such membrane potential fluctuations between a hyperpolarized down - state and a depolarized up - state , which may gate the information and gain modulation .
here , we test the gain change of ra projection neurons after exogenous nmda was applied to selectively activate nmda receptors , using whole - cell current clamp recording in adult male zebra finch slices .
brain slices were prepared from adult male zebra finches ( taeniopygia guttata ) ( > 90 days old ) obtained from commercial suppliers as previously described [ 19 , 20 ] .
brains were dissected into ice - cold , oxygenated ( 95% o2 and 5% co2 ) slice solution .
slice solution consists of ( in mm ) sucrose 248 , kcl 5 , nahco3   28 , glucose 10 , mgso47h2o 1.3 , and nah2po4h2o 1.26 [ 21 , 22 ] .
coronal brain slices ( 250  m thick ) containing ra were cut with a vibrating microtome ( ma752 , usa ) and collected in artificial cerebrospinal fluid ( acsf ) that had been warmed to 37c and subsequently allowed to cool to room temperature .
slices were allowed to recover in the holding chamber for at least 1.5  h and were equilibrated to room temperature before recordings were made .
standard acsf consists of ( in mm ) nacl 125 , nahco3 25 , nah2po4h2o 1.27 , kcl 2.5 , mgso47h2o 1.2 , cacl2   2.0 , and glucose 25 and was adjusted with sucrose to a final osmolarity of 350  mos . for recording ,
a slice was transferred to a submerge - type chamber where it was continuously exposed to acsf , saturated with 95% o2 and 5% co2 at room temperature ( 2226c ) at the rate of 2.0  ml / min .
ra and the surrounding tissues were distinguishable under bx51wi microscope connected with a dic - ir video camera ( olympus , japan ) . at high magnification ( 40x ) , ra neurons were visualized and the recordings were made from healthy cells .
recording pipettes were fabricated from borosilicate glass ( sutter instruments , usa ) using a flaming - brown puller ( model p-97 , sutter instruments , usa ) and were filled with the pipette solution containing ( in mm ) : k meso4   120 , nacl 5 , hepes 10 , egta 2 , mg - atp 2 , and na3-gtp 0.3 ( ph 7.3 - 7.4 , and 340  mos ) .
the recording pipettes , which had resistances ranging from 3 to 5  m , were positioned using an integrated motorized control system ( sutter instruments , usa ) .
the potential changes after application of nmda were corrected during the input - output stimulation protocols .
the series resistance ( rs ) , between 15  m and 30  m , was compensated using the bridge balance .
all agents were applied by changing the bath perfusate from standard acsf to modified acsf in which various drugs were simply added . unless indicated otherwise ,
the clampfit 9.2 ( axon instruments , usa ) , mini 6.0 ( synaptosoft inc . , fort lee , nj , usa ) , and originpro 8.0 ( originlab , nothampton , ma , usa ) were used for analysis .
events with peak amplitude of 50  mv or higher and a rise time of about 0.5  ms were detected automatically , and the results were analyzed with origin pro 8.0 .
the steady - state spike rate was estimated by counting the number of spikes in 1 second , and the result was plotted versus the intensity of the injected current ( f - i relationship ) .
the slope of the f - i relationship ( referred to as gain ) was estimated by linear fitting .
slope parameters were estimated separately for individual neurons and that figures 4 and 5 contain mean slope values averaged for the whole groups of neurons .
the amplitude of ahp was quantified as the difference between spike threshold and the lowest point of ahp . throughout
means were compared using paired two - tailed student 's t - test or one - way anova followed by scheffe 's multiple comparison .
stable whole - cell recordings were obtained in 160  ra projection neurons from 51 adult male zebra finches .
a neuron with the resting membrane potential < 50  mv and an overshooting action potential was considered viable .
ra projection neuron was identified by a larger soma , time - dependent inward rectifier induced by hyperpolarizing current and spontaneous regular spike firing or by depolarizing current pulses .
the input - output relationship was examined by injecting current pulses from 0 to 90  pa with an increment of 10  pa and an interval of 15  s , and they were lasted for 1  s. traces of action potentials in response to 1  s current steps of 10 , 50 , and 90  pa were shown in figure 1(a ) .
the slopes of the f - i relationships were 246.92  28.00  hz / na before nmda application and increased to 325.49  73.34 
furthermore , the input resistances were decreased by 20  m nmda in ra projection neurons ( p < 0.05 , n = 6 ) ( figure 2 ) .
the afterhyperpolarization ( ahp ) of action potential underlies the modulation of firing frequency . to test the effect of nmda on spike ahp
comparison of spike shape is shown in figure 3(a ) with 90  pa current injection before and after nmda application . in average
, there was no significant change in the amplitude of ahp after nmda application ( p > 0.05 , n = 5 ) ( figure 3(b ) ) .
then we used 50  m dl - apv , a nmda receptor antagonist , to check the selectivity of nmda receptor , and we found that the slope of f - i was not changed by dl - apv alone ( p > 0.05 ) .
the slope was 255.78  66.69  hz / na and 269.34  56.96  hz / na ( p > 0.05 ) before and after application of 50  m dl - apv ( figure 4 ) , respectively .
nmda increased the f - i slope 1.32 times ( p < 0.05 ) ; however , the gain modulation induced by nmda was completely abolished by dl - apv ( figure 4 ) .
previous studies reported that ra projection neurons received glutamatergic and gabaergic inputs [ 23 , 24 ] , which might increase background synaptic activities and modulate the gain of neurons [ 25 , 26 ] . to determine the effect of ampa receptor and gaba receptor on gain modulation by nmda , we examined the effects of nmda in the presence of cnqx , an antagonist for ionotropic glutamate ampa receptors , or / and bicuculline methiodide ( bic ) , a competitive antagonist of gabaa receptors , respectively .
bic ( 10  m ) accelerated the depolarization by nmda application , but bic alone had little effect on the f - i slope . in the presence of bic ,
the mean initial slopes were 276.46  59.4 and 332.6  40.02  hz / na before and during application of nmda .
the f - i slope was increased from 1.32 to 1.39 in the presence of nmda and bic ( p > 0.05 ) ; that is , there is an increase of 105.30% over the nmda treatment ( figure 5 ; p > 0.05 , unpaired t - test ) .
cnqx ( 10  m ) decreased the membrane potential and abolished the spontaneous spikes of ra projection neurons sometimes but did not affect the slope of f - i relationship . in the presence of cnqx ,
nmda application hardly changed the f - i slope of ra projection neurons no matter whether bic was present or not ( figure 5 ; p > 0.05 ) .
these results indicated that the ampa receptor - dependent excitability played a crucial role in the gain modulation by nmda .
the slope was 0.97 or 1.11 in the presence of cnqx or cnqx and bic , respectively .
the gain induced by nmda was significantly decreased by cnqx ( p < 0.05 ) .
in this study , we showed that nmda increased the gain of ra projection neurons in adult zebra finch .
nmda - induced gain increase was limited to firing frequencies less than 40  hz . the effect of nmda on gain was mediated through nmda receptors and might involve synaptic activities . in songbirds , we found that nmda increased the slope of f - i relationship in ra projection neurons , which was completely blocked by dl - apv , a nmda receptor selective antagonist , suggesting that nmda increased the sensitivity of ra projection neurons .
the pharmacology of nmda receptors involved in gain modulation has been examined in mammals . in cat
visual cortex , nmda increased the gain of the contrast - response ( c - r ) curve of the neuron in layers ii / iii and v / vi . in mouse , the thalamocortical neurons in the dorsal lateral geniculate nucleus play a key role in the generation of firing patterns through nmda receptors .
the neural activity in the afp of songbirds can direct moment - by - moment changes in the primary motor areas responsible for generating song .
singing - related activity in lman is more variable in pattern across repeated renditions during undirected song than directed song [ 28 , 29 ] .
reported that the fundamental frequency of the syllable was shifted by stimulating the lman - ra pathway , which is mediated by nmda receptors .
our results indicated that the activity of nmda receptor changed the sensitivity of ra projection neuron .
ra , as a premotor nucleus , occupies an important position in the song system , integrating information from both pathways .
ra projection neurons are responsible for transmitting song information to midbrain and brainstem vocal and respiratory structures . temporal pattern of spike bursts in ra projection neurons is associated with the timing of acoustic features of birdsong .
precise timing of individual spikes has a close relationship with stereotypic behavior , which suggests that the song is represented in ra by a temporal code [ 5 , 31 ] .
thus , the modulation of nmda receptors in the activities of ra projection neurons could be very important for the plasticity of birdsong .
previous studies in slices demonstrated that nmda receptor achieves the nonlinear , multiplicative behavior seen in gain modulation by several mechanisms .
mel presented results from compartmental modeling studies showing that dendrites containing nmda - type synaptic conductances and voltage - dependent na , k , and ca channels can impose nonlinear interactions between nearby synapses that give rise to approximate multiplications [ 32 , 33 ] . in thalamocortical cell of the rat and cat dlgn , nmda receptors contribute to the burst firing generated by the low - threshold ca potential and depend on the membrane potential range over which this type of firing occurs .
in substantia nigra dopamine neurons , nmda receptor - mediated synaptic conductance generates transient high - frequency activity by rapidly but transiently overwhelming the conductance 's underlying action potential ahp and/or engaging postsynaptic voltage - dependent ion channels in a manner that overcomes the limiting effects of ahp .
but our results showed that there was no significant change in spike ahp after nmda application .
so the gain induced by nmda in ra projection neurons may not be mainly modulated by the change of spike shape , but by synaptic activities .
the change in membrane potential has been shown to play an important role in the firing pattern regulation by nmda receptor , supporting the idea that depolarization acutely alters firing frequency .
it has been shown that simple excitability or inhibition alone can induce a multiplicative gain change in the cortical neurons . in this model ,
one set of synaptic inputs provided purely excitatory drive to a target neuron , and another set of combined excitatory / inhibitory inputs ( or balanced inputs ) provided the modulation . in this setup , changing the firing rates of the balanced inputs produced an approximately multiplicative gain modulation of the response to the purely excitatory drive . here
, blockade of the excitatory or inhibitory transmission alone had no effect on the f - i slope of ra projection neuron , implying that simple excitability or inhibition alone can not induce a multiplicative gain change .
however , our results did not exclude a role of spontaneous synaptic activity on gain modulation in vitro , which , due to the loss of synapses in slice , was not sufficient to produce a significant change in gain .  
ampa receptors are required for the gain modulation in our study , because blocking excitatory transmission mediated by ampa receptors almost abolished the effect of nmda on gain modulation .
two reasons might be involved : one is the two states of nmda - base mechanism due to the special anatomical structure of ra .
ra projection neurons receive inputs from two areas , hvc and lman . both are excitatory glutamatergic but have distinct postsynaptic properties .
hvc - ra is largely mediated by ampa receptor , whereas lman - ra is almost completely mediated by nmda receptor .
ampa / nmda receptor composition of different neuronal pathways is the foundation of gating and gain modulation .
nmda receptor - rich pathway can gate ampa - rich input and increase the gain of a neuron responding to the input of ampa - rich pathway .
the second is related to the intrinsic properties of nmda receptors , which are uniquely voltage dependent . except binding with neurotransmitter
blockade of main excitatory inputs mediated by ampa receptor with cnqx hyperpolarized the membrane potential , which caused nmda receptors not activated .
therefore , the ampa receptor - mediated excitatory transmission is essential for the gain modulation induced by nmda .
it is well known that the nmda receptor plays a critical role in many physiological and pathological processes , such as learning , memory , neurodegeneration , epilepsy , and ischemia .
the nmda receptor - mediated synaptic transmission satisfies the associative property of hebbian learning and in fact plays a critical role in its cellular model , long - term synaptic plasticity .
nevertheless , pharmacological agents which block nmda receptors impair a variety of brain processes , suggesting that nmda receptor transmission also plays an important role beyond long - term memory and participates in shaping the dynamic activity of neural networks . in song system
, the expression pattern of nmda receptor is related to the development of song learning [ 21 , 3740 ] .
recent results show that the nmda receptor on the lman - ra synapses generates variability across different song renditions , thereby facilitating reinforced learning of songs . how nmda accomplishes this role is not clear .
our results showed that nmda application increased the gain of ra projection neurons , in which ampa receptor was necessary .
the ampa receptor - mediated excitability is derived only from hvc [ 10 , 11 ] .
therefore , nmda receptor in lman - ra synapse might regulate hvc - ra synaptic transmission , which implicated that nmda receptor is more effective to modulate the syllable online .
these findings suggest a mechanism by which nmda receptor can selectively modulate behaviorally relevant excitatory inputs .
 the song of zebra finch is stable in life after it was learned successfully . 
 vocal plasticity is thought to be a motor exploration that can support continuous learning and optimization of performance . 
 the activity of ra , an important pre - motor nucleus in songbird 's brain , influences the song directly . 
 this variability in adult birdsong is associated with the activity of nmda receptors in lman - ra synapses , but the detailed mechanism is unclear . 
 the control of gain refers to modulation of a neuron 's responsiveness to input and is critically important for normal sensory , cognitive , and motor functions . here , we observed the change of gain in ra projection neurons after exogenous nmda was applied to activate nmda receptors using the whole - cell current clamp recording . 
 we found that nmda substantially increased the slope ( gain ) of the firing rate - current relationship in ra projection neurons . 
 the ampa receptor - dependent excitability played a crucial role in the modulation of gain by nmda . 
 these results suggested that nmda receptors may regulate the dynamics of ra projection neurons by input - output gain . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion