Temperature manipulation of neuronal dynamics in a forebrain motor control nucleus
[['Coordinated motor behaviors require a delicate sequence of gestures .', 'Their precise timing depends on instructions coming from motor control areas in the brain .', 'Differentiated regions or nuclei are interconnected forming a motor pathway .', 'However , little is known about how the detailed motor program arises from the dynamics of individual neurons .', 'A widely used model to study complex behavior is birdsong , which consists of a succession of highly stereotyped vocal gestures .', 'Like humans with their speech , oscine birds that account for approximately forty percent of the known species , need to learn their vocalizations from a tutor [1–5] .', 'The well characterized set of forebrain nuclei in charge of this task is called the “song system” [6–8] .', 'It has proven to be a valuable model to study the network properties necessary to generate the precise motor patterns of song [9–12] .', 'Motor nucleus HVC ( proper name ) appears to have a key role in song production .', 'Important aspects such as the structure and sequence of the different syllables that compose songs are represented in the activity of its neurons [13] .', 'The representation of time and of important events in motor gestures have been described in this nucleus [14–17] , as well as a proposed role in sensorimotor integration [12 , 18 , 19] .', 'Continuous temporal encoding of vocal features was proposed as a key role of HVC [16 , 20] .', 'However , this view has been open to debate recently with the proposition that activity in this nucleus only encodes relevant instances of song [17 , 21–23] , and by a complementary proposition [24 , 25] in which song timescales appear from the interaction of the HVC with the rest of the motor pathway .', 'These have broadened the discussion regarding the possible motor coding of song in HVC , leading to the proposition of recurrent networks for song timing generation [23 , 26–29] .', 'In addition , recently , it has been shown that HVC projecting neurons fire densely during singing behavior [30–32] , and that another nucleus in the song pathway , the subthalamic nucleus Uva , has a significant contribution to song timing [33 , 34] .', 'It remains an open and exciting field for further research since it is still lacking a definitive consensus for the precise role of HVC on the timing of birdsong .', 'At a smaller scale , how individual neurons in HVC contribute to generate the song sequence is also not fully understood .', 'They project both to the forebrain nucleus RA ( robust nucleus of the arcopallium ) and to a basal ganglia nucleus , area X , which is essential for song learning and belongs to the anterior forebrain pathway [35] .', 'For some time , the vocal motor pathway was believed to have a top-down architecture , initiating activity in HVC , projecting to RA which in turn projects to the respiratory center ( RAm and PAm , nucleus retroambigualis and parambigualis ) and to the motor nucleus innervating the vocal organ ( nXIIts ) .', 'However , experiments show that there is also a bottom-up architecture that goes from the brainstem back to HVC that influences the activity of HVC neurons [27 , 34 , 36–38] .', 'A novel technique that has been developed recently to manipulate activity within HVC is local temperature control [16 , 24 , 25 , 33 , 39–41] .', 'It was shown for zebra finches ( Taeniogypia guttata ) that cooling HVC leads to a stretching of the song tempo without major changes in the acoustic structure of song [16 , 39] , and for canaries ( Serinus canaria ) an initial stretching and a restructuring of song , or “syllable breaking” for colder temperatures [24] , and the possibility of controlling syllable transitions [25 , 40] .', 'The working hypothesis for these experiments is that a timescale represented in HVC is being manipulated with temperature , somehow making it slower at colder temperatures .', 'Recent work in zebra finches also cooled Uva to show that song timing control is distributed in a recurrent network [33] .', 'The individual neuronal mechanisms that take part in these physiological observations within HVC will be studied in this work .', 'Previous in vivo studies of the relationship of neuronal functionality and temperature were mostly done in poikilothermal animals , which are cold blooded and cannot regulate efficiently their temperature .', 'The focus on those studies is in understanding how function can be robust and resilient to large temperature fluctuations of the brain [42 , 43] .', 'However , many neuronal properties studied in these animals provide evidence of widespread mechanisms .', 'The main findings point to timing changes with temperature decrease: period and burst interval decrease in acoustic [44] and electric [45] communication in fish .', 'Acoustic perception seems to be deteriorated at lower temperatures in locust [46 , 47] , as spiking variability increases with lower rates , the same effect that is present in the fly vision system [48] .', 'Another widely studied model is the crab stomatogastric system , in which a central pattern generator , although increasing burst period and inner frequency , is able to compensate its phase over a wide range of temperatures [49 , 50] .', 'Also compensation is found in the bullfrog respiratory rhythm , whereas some neurons paradoxically increase firing at lower temperatures [51] .', 'Few studies were done in homeothermal animals apart from the recent birdsong focal cooling .', 'These divide into ones that monitor the temperature of the brain and others that use slices and modify the temperature of the bath of the recording solution .', 'Brain temperature can fluctuate up to 2°C when animals do exercise [52] , when they are presented a female [53–55] and when they are given cocaine [56] .', 'Temperature correlates with neuronal properties like latency after electrical stimulation and dynamics of EPSPs , as was shown in rat hippocampus [52] or song tempo , as shown in HVC in zebra finches [55] .', 'Also resting membrane voltage change is usually reported as it is believed that it can put the neuron closer or further to the spiking threshold .', 'Although many report an increase in this value at low temperatures ( 4mV in hippocampus [57] , 15 mV in rat visual cortex [58] with a 10°C change ) , others report decreases ( 10mV in one type of hippocampus interneurons [59] ) .', 'Finally , cooling has also been used as a tool to change perception of interval timing in rat prefrontal cortex [60] .', 'In all these studies , there is also emphasis in the reversibility of these effects when temperature is returned to normal .', 'The studies mentioned above highlight the increase in timescales and decrease of level of activity present in neuronal behavior with temperature decrease , and that these come from changes in rate and conductance constants .', 'In the other hand detailed modeling of temperature effects has not been explored extensively .', 'The work done in the stomatogastric ganglion of the crab is the most comprehensive , showing a full exploration of the parameter space of extrapolation constants for each ionic rate and conductance [43 , 61] .', 'Just above 5% of the hundreds of thousands of models explored showed bursting activity , and only 0 . 5% showed the robust behavior measured experimentally , although being more than 500 different possible parameter combinations .', 'In our case of study , there are three classes of neuronal populations in HVC: excitatory neurons projecting to nucleus RA and X ( HVCRA and HVCX respectively ) , and inhibitory interneurons ( HVCINT ) [62 , 63] .', 'Intracellular recordings in vitro and in vivo helped to reveal neuronal and circuit mechanisms , and physiological properties of these neurons [63–67] , and the connectivity between them was studied by means of paired intracellular recordings and antidromic stimulation in slices or by means of retrograde labeling [28 , 64] .', 'The recent work by Daou et al . has described extensively the ionic currents present in each neuronal type with an exquisite match between electrophysiological measurements and computational single compartment models using different stimulation protocols in brain slices [67] .', 'Recent single cell modeling in HVC is based in these findings for excitatory neurons [68] , and also was upgraded to two compartments for HVCRA [69] .', 'Network models have also being explored , but less is known about the specific connectivity within HVC [66 , 70 , 71] .', 'In this work we study electrophysiologically the neuronal in vivo mechanisms that manifest in HVC neurons of canaries when the nucleus temperature is decreased locally .', 'We took advantage of the very precise neuronal models developed by intracellular studies in vitro .', 'Although intrinsic parameters were obtained at room temperature , we could extrapolate them to the in vivo temperatures used in this work .', 'We found that the three most characteristic changes with temperature of the experimental neuronal dynamics can be explained with the computational model .', 'We also make predictions about connectivity and possible experimental implementations .'], ['All experiments were approved by the Institutional Animal Care and Use Committee of the University of Buenos Aires .', 'Adult canaries were acquired from a local breeder .', 'The experiment was performed on 11 male canaries ( Serinus canaria ) either on the left HVC ( N = 6 ) or the right HVC ( N = 5 ) .', 'The morning of the experiment , birds were deprived from food and water 45 minutes prior to surgery .', 'They were given injections in the pectoral muscle with 20% urethane ( 90–120μl total; Sigma , St . Louis , MO ) , administered in three 30–40μl doses at 30 minutes intervals .', 'Immobilization of the bird was done with a stereotactic device .', 'After topical application of xylocaine , the skull of the birds was exposed by sculp retraction .', 'A craniotomy of 2mm x 1mm was made over the corresponding left or right HVC which was located stereotactically and the dura over the recording site was removed .', 'Body temperature was maintained with an electric blanket .', 'Two custom built cooling devices were used , one for placing over the left and the other over the right HVC .', 'Their capabilities are based on a Peltier module , which provides a constant temperature decrease on one side while the other is kept at a constant temperature .', 'Previously , a 2A current supply was used for obtaining a temperature decrease of 10°C [16 , 24] .', 'Such high currents circulating continuously near the recording site interfere with the electrophysiological signal about a minute after onset .', 'To avoid this we increased the Peltier size four times and performed a pulsed current protocol with a period of 6 seconds ( S1 Fig ) .', 'To avoid neuronal damage due to abrupt temperature changes , currents were modified in steps of 0 . 25A and the temperature was allowed to stabilize for at least 3 minutes .', 'We achieved almost constant fluctuating temperatures with a maximum decrease of approximately ΔT = −9°C and a maximum amplitude of fluctuation of less than 0 . 5°C .', 'Calibrations of the devices were made on two supplementary animals not used for the electrophysiological recordings ( S1 Fig ) .', 'We obtained between 4 and 10 different temperatures for 33 different recording sites with an average of 8 . 7 temperatures ( 30 sites with 7 or more temperatures ) .', 'To rule out the possibility of permanent effects due to cooling , we made half the measurements increasing the current and then half going back to 0A .', 'A metal part was designed to allow the most efficient heat transfer between HVC and the cooling device ( S1 Fig ) .', 'It has a pad of 1mm x 2mm surface and 1mm thickness that is placed on the surface of the brain right above HVC with a 0 . 5 mm diameter hole made in its center .', 'Through that orifice we lowered the measuring electrode .', 'Extracellular recordings of spontaneous neural activity in HVC were performed using tungsten microelectrodes ( Parylene-C-insulated , 0 . 8-1 . 2MΩ , MicroProbes ) .', 'The electrodes were lowered between 200 and 800 μm beneath the surface of the brain , because HVC is approximately 1mm thick and is located superficially .', 'Electrode entrance in HVC was identified by its neuron’s characteristic spontaneous bursting patterns .', 'Signals were preamplified 10X near the electrode via a custom built , low power , low noise and battery powered instrumental amplifier [72] .', 'Then this signal was fed into an Instrumentation Amplifier And Signal Conditioner ( Brownlee Precision Model 440 ) , where it was further amplified 100X , bandpass filtered between 300 Hz and 3 kHz and digitized at 20 kHz .', 'The final output was partitioned in 5 to 10 contiguous files and stored on a computer through a data acquisition device ( National Instruments M Series ) .', 'Total recording times for each site range from 2 to 8 minutes ( 4 . 8 min in average , 31 sites above 4 min ) .', 'We implemented single-compartment conductance-based biophysical models of neurons from the HVC following the ones developed by Daou et al . [67] .', 'Simulations were performed in C using a Runge Kutta 4 algorithm for integration with 0 . 01ms steps , and the analysis of the model output was made in MATLAB .', 'Models are of Hodgkin-Huxley-type with additional currents .', 'The membrane potential of the neuron obeys the following equation:', 'C m d V d t = - I L - I K - I N a - I C a - L - I C a - T - I A - I S K - I K N a - I h - I N a p - I s y n + I a p p + I n o i s e ( 1 )', 'where Cm is the membrane capacitance , IK and INa are spike-producing currents , ICa−L and ICa−T are Ca2+ high-threshold L-type , and low threshold T-type currents , ISK is a small-conductance Ca2+-activated K+ current , INap is a persistent Na+ current , IKNa is a Na+-dependent K+ current , IA is an A-type K+ current , Ih is a hyperpolarization-activated cation current , and IL is a leak current .', 'All these ionic currents were found experimentally in slice electrophysiology experiments at room temperature in all three HVC neuronal types and detailed equations and parameters can be found in Daou et al . [67] .', 'Here we briefly comment on the different currents whose relative weight change across neuron types , shaping their characteristic behavior at room temperature: HVCX neurons show spike adaptation due to large ISK and IKNa currents and have a sag due to Ih current .', 'They show rebound firing or depolarization due to large ICa−T current .', 'HVCRA neurons show lack of excitability in response to depolarizing pulses due to large ISK , IKNa and IA currents , and a delay to spiking due to large IA current .', 'HVCINT neurons show high firing frequency with no adaptation even to small pulses due to reduced ISK , IKNa and IA , a prominent sag and hence a rebound firing due to large Ih current and also fire spontaneously with variety of patterns in slice preparations .', 'Sodium INa and potassium IK also vary among neuron types .', 'The most interesting and novel observation in this description is the role that ISK may play in the bursting behavior of HVCRA and HVCX in vivo , having a slow current dependent on the dynamics of the free intracellular Ca2+ concentration .', 'Although bursting did not appear at room temperature , experiments in vivo where the temperature is around 40°C show clearly their bursting behavior [66] .', 'A typical current is as follows:', 'I c = g c x ( V - V c ) ( 2 )', 'where Ic is any of the currents described before , gc its maximal conductance , Vc its reversal potential and x is a gating variable whose kinetics are governed by:', 'd x d t = x ∞ ( V ) - x τ x ( 3 )', 'where x∞ ( V ) is a defined function of voltage and τx is the time constant , with 1/τx a factor that multiplies the rate constants ( usually called k values ) of opening and closing of the ion channel which for some currents has a membrane potential dependency τ ( V ) .', 'For different simulations we used artificially applied external currents Iapp , synaptic currents Isyn and some degree of current noise Inoise .', 'The first is just a current clamp: constant input with a finite duration .', 'The second needs to have a presynaptic neuron releasing neurotransmitter T . Equations used read as follows [75 , 76]:', 'T ( t ) = T m a x 1 + e x p - ( V p r e - θ ) σ ( 4 ) d s d t = α T ( t ) ( 1 - s ) - β s ( 5 )', 'I s y n = g s y n s ( V - V r e v ) ( 6 )', 'where a T ( t ) approximates a Heaviside function that turns on with the voltage of the presynaptic neuron Vpre .', 'Parameters θX = −10mV , θRA = −20mV and σX = 7mV σRA = 5mV were selected after evaluating the amplitude and maximum voltage achieved by HVCX and HVCRA simulations in order to have neurotransmitter release at approximately the top 10% of the simulated presynaptic spikes .', 'The rate constants of the gating variable s , α = 2 . 2mM/ms and β = 0 . 381/ms , and the maximal neurotransmitter concentration Tmax = 1 . 5mM were selected as in Gibb et al . [70] .', 'Typical values for Vrev are 0mv for excitatory synapses and -80mV to -100mV for inhibitory synapses , and here we modeled only excitatory synapses .', 'gsyn = 3 . 0nS and 2 . 5nS for HVCX and HVCRA were selected to have slightly less than 100% effectiveness in eliciting a spiking response in an interneuron when noise is present and to be able to reproduce the in vivo recorded firing pattern at a single excitatory synapse as recorded by Kosche et al . [28] .', 'We added to some simulations standard Gaussian white noise:', 'I n o i s e = n I a m p ( 7 )', 'where n is a random number drawn at each time step from a normal distribution and its amplitude Iamp = 400pA .', 'It was changed in 0 . 1ms steps .', 'Temperature control in the model was made by modifying the electrical properties most strongly affected by temperature: the maximal conductances and the rates of channel opening and closing [77] .', 'To add these effects to the model we changed conductances gc and time constants τx , using the usual Q10 based formalism:', 'p = p r e f Q ( 8 ) Q = Q 10 ( T - T r e f ) / 10 ( 9 )', 'where the parameter p is a conductance g or a rate constant 1/τ , Tref is the temperature where the parameters were originally fitted with the model from measurements , Q is the scaling factor , T is the temperature and Q10 describes temperature sensitivity .', 'Typical values for Q10 are Q 10 k = 3 for rate constants and Q 10 g between 1 . 5 and 3 for conductances [49 , 50 , 61 , 77] .', 'Exploration was made in these parameters with steps of 0 . 1 from 1 . 1 to 3 . 2 to obtain the best match between experimental and computational spike traces of each HVC neuron .', 'We assumed a value of Tref = 20°C which is mentioned by Daou et al . [67] as “room temperature” , however values up to 25°C do not modify our results .', 'Temperature exploration with parameter T was made between 40°C and 20°C , with the former being the designated “normal” temperature of the bird’s brain .', 'Other explorations were made in the range 40 − 30°C , the latter being almost the lowest temperature obtained experimentally .', 'We also changed temperature in ICa−L that has an explicit T dependence in Kelvin units .'], ['We first classified the SUs by their waveform shape .', 'In Fig 1A we can see all the mean waveforms normalized and aligned to their lowest value .', 'We could separate them into two groups by the mean of their peak to peak width .', 'Widths that were less than 0 . 5ms were classified as Fast Spikers ( FS ) and the longer ones as Regular Spikers ( RS ) .', 'Usually these shapes are related to the neuronal type , with the former classified usually as interneurons , while the latter are usually designated as excitatory neurons ( Fig 1B ) .', 'The first easily visualized effect found in all neurons , was the spike shape widening of the extracellular potential ( Fig 1C ) as temperature decreased from normal at 40°C down to almost 31°C .', 'Single units were isolated at each measurement site by means of spike detection and clustering techniques ( See Methods ) .', 'Mean waveforms were obtained after aligning all the events to the minimum voltage , and spike times were used to quantify activity and inter-spike-interval ( ISI ) distribution characteristics .', 'In Fig 1C we can appreciate five widening examples , three FS and two RS .', 'The widening effect reached up to almost a 2-fold increase from the original shape for FS .', 'To properly assess the effect for the regular spikers , we pulled out from the 18 cell measured the 5 highest firing ( RShf ) and the five lowest firing ( RSlf ) in an attempt to represent HVCX and HVCRA respectively .', 'The rationale for this is the following .', 'Since the wave shapes obtained from extracellular recordings of both types of cells are indistinguishable , we took advantage of the differences in their spike rate activity .', 'Assuming that the probability of measuring any of both cells is equally probable , taking the lowest 5 and 5 highest firing cells gives a value p< 0 . 05 ( p = 0 . 048 ) of not having selected all from the same type .', 'This is given by the binomial cumulative density function at a value of 5 with 18 samples .', 'This selection will prove to be appropriate after the delicate match with the model that we show below .', 'The widening effect was lower in RS units than in FS units , and it was more pronounced in RShf compared to RSlf , as we can see in Fig 1C .', 'The increase was of 40% and 20% respectively ( Fig 1D , S2A Fig shows not normalized values ) .', 'In addition to the widening effect , we could see that the activity decreased substantially in all cases with temperature with no significant difference between cell types ( Fig 1E , not normalized values in S2B Fig ) .', 'However , the way that the activity changed differed for each firing pattern .', 'In six of the FS units , most of the activity above an ISI of 10ms gradually disappeared with lower temperatures , retaining many occurrences below 10ms , as we can see in first column of Fig 1F ( neuron FS 1 ) .', 'Other behavior observed in two FS units is depicted in the second column of Fig 1F ( neuron FS 2 ) .', 'These neurons do not present bursts at normal temperature , and have ISIs which peak at around 20ms .', 'As temperature decreases the distribution of ISIs depletes and shifts to the right .', 'Finally , there were four neurons that did not present the longer timescale , and although decreasing the firing rate , they did not show changes in the ISI distributions ( Fig 1F , neuron FS 3 ) .', 'The existence of different firing behaviors of HVCINT ( FS ) was previously shown by Daou et al . [67] .', 'The quantification of these two timescales and the disappearance of the second one is made evident with the cumulative distribution functions shown in Fig 1G .', 'Second timescale disappears in neuron FS 1 and neuron FS 2 , but not in neuron FS 3 where it was not originally present .', 'To better quantify these timescales we did separate analyses below and above 20ms , which showed that median values in these ranges have different correlation with temperature , rising for above and decreasing for below 20ms ( S2C and S2D Fig ) .', 'This implies that although having an almost 3 fold reduction in firing rate in the range of temperatures studied , the timescale appearing below 10ms does not disappear ( for the neurons that have it ) , and that the timescale around 20ms disappears with decreasing temperatures .', 'In addition , only the neurons having the intermediate timescale showed differences with the cumulative distribution at normal temperature ( S2E–S2H Fig ) revealing non trivial modifications of the ISI distributions .', 'For the two example RS units shown in Fig 1C , the evolution of the ISI distribution retains its structure , but loses it at times higher than 20ms .', 'This is clearly seen in the third row of the last column of Fig 1F ( RS lf ) , where the second peak in the distribution disappears .', 'In all cases we can see that the first peak or left end of the distribution moves slightly to the right , to a higher ISI .', 'We did not find differences in all these effects between the left and right HVC .', 'In what follows , we will study with the help of a computational model of the neurons in HVC , the possible explanations for the temperature effects in the spontaneous activity of its neurons .', 'Computational modeling of HVC neurons has proven to be successful for in vitro approaches [67] .', 'In those studies , single compartment conductance descriptions of currents and voltages were obtained with great detail .', 'Further work also explored non conventional complex input currents designed to activate most ionic channels and data assimilation procedures to better evaluate parameters [68] .', 'These works show astonishing match between experiment and modeling , making them ideal to test temperature extrapolations .', 'As mentioned above , the works done in the crab made a full exploration of the parameter space showing that a very small percentage of models will show the expected physiological behavior , but that they are nevertheless hundreds of parameter combination possibilities .', 'This emphasizes that many models can be equivalent to explain a behavior , and that care has to be taken in restricting constraints .', 'Other modeling variations of HVC neurons include two compartments , soma and dendrites [66 , 69] , to better explain the absence of bursting from current injections in the soma of HVCRA neurons in slices at 35°C [66] .', 'However , dynamics of bursting within a single compartment neuron are expected to not differ from a two compartment one apart from a small delay coming from the electric couping constant between the two compartments ( not more than ∼1ms ) .', 'Current injections in the soma will elicit bursting since the IK Ca dependent current mostly responsible for it is located in the same compartment as the currents responsible for spikes .', 'In our modeling we will use the parameters found by Daou et al . and a single compartment model .', 'Our hypothesis is that there is a parameter range were the responses of our model are robust to changes .', 'Therefore we selected the extrapolation parameters using constraints from previous in vivo studies .', 'Our first step was then , starting from these previous results , to extrapolate them to in vivo brain temperatures .', 'As is usual in most of the in vitro electrophysiological studies , temperature is mentioned as “room temperature” .', 'This fact has consequences , like further studies in the field yielding results that differ from the previous ones , since they work at another “room temperature” .', 'For the purposes of this work we did center our interest in dynamical regimes , rather than in absolute values , since they are expected as we showed in the previous section in the almost 10°C temperature range explored .', 'Our studies focus in the global changes manifested and not the exact temperature at which they occur .', 'Therefore we decided arbitrarily to assign a 20°C value to the temperature where the computational parameters were obtained .', 'As explained in the Methods section , we modified the maximal conductances and the rates of channel opening and closing based on an already successful temperature model for neuronal activity [43 , 49 , 50 , 61] .', 'Search in parameter space was done with the premise that different Q10 values act on the maximal conductances and the rate variables , and the search for the proper parameters was done independently for the three neuronal types .', 'We aimed at reproducing previous intracellular recordings that show bursting in excitatory cells and their specific voltage evolution [63 , 66] .', 'We decided to not separate each of the specific Q10 values for gating and conductance variables ( 7 for rates and 10 for conductances ) , since finding a combination that reproduces the expected behavior should be robust under slight changes in the parameters [43] .', 'Therefore , we made a two dimensional parameter search with the two Q10 parameters decoupled for the three neuronal types .', 'Q10 k ( rate ) and Q10 g ( conductance ) were allowed to vary from 1 . 1 to 3 . 2 in since 1 means no effect by temperatures , and 3 is the usually biggest reported value .', 'We explored the temperature range from T0 = 20°C to the normal temperature of 40°C .', 'We injected 16ms length currents with 3 varying steps .', 'Criteria for selecting parameters was capability of bursting and number of spikes and measures of voltages between resting , maxima , minima and local minima .', 'Finally , we assessed resemblance with voltage traces from the literature .', 'First , criteria needed to be met at 40°C and then we checked for robustness across other temperatures .', 'The easiest search was made in HVCRA , because it presents a narrow range of parameters where it presents bursting ( Fig 2A ) .', 'The selected value is ( Q10 k , Q10 g ) = ( 3 . 0 , 1 . 4 ) .', 'Second , HVCINT ( Fig 2B–2D ) presents strong spiking at all parameters , and a region with strong firing and robustness across temperature was selected .', 'Then we looked for the difference between minimum hyperpolarization after spike and resting membrane potential , as minimum difference was found in in vivo studies [63 , 66] .', 'As a last constraint , we selected a region were the peak to peak voltage was neither too high neither too low and about 60-80 mV .', 'Values for the interneuron are ( Q10 k , Q10 g ) = ( 3 . 0 , 2 . 5 ) .', 'Third , we studied the behavior of HVCX which needed further constraints to arrive to the selected values ( Fig 1E–1H ) .', 'Higher and more robust spiking across temperatures was found first and then we assessed interburst interval to be lower than 4ms .', 'Peak to peak voltage inside a burst was chosen to be between 40 and 60mV , because we know it spans from about half to 75% of its 80mV peak to resting membrane amplitude .', 'As a last measure , we evaluated the peak to peak width to be bigger than 0 . 6ms , as suggested by our measurements ( see S2A Fig ) , and to be smaller than 2ms which is about the half of the interburst interval .', 'Parameters chosen from the intersection of selected zones are ( Q10 k , Q10 g ) = ( 3 . 0 , 2 , 5 ) .', 'We did also explore other parameters as the resting membrane potential ( S3 Fig ) .', 'The only neuron to show a variability bigger than 1mV across all the parameter space and from 20 to 40°C was HVCX .', 'In that case , the variation in the range of experimental temperatures studied was of almost 4mV , and it increased at low temperatures .', 'The other neuronal types also showed this increase , although it is of less than 1mV .', 'This behavior is compatible with the measurements on hippocampal slices mentioned above .', 'The obtained Q 10 k = 3 factor for rate constants was the same for the three neuronal types , and the evolution of Q with temperature can be seen in Fig 3A .', 'It represents probability of channel opening and closing effects and is the typical value measured in the bibliography and used in models [49] .', 'On the contrary , the temperature sensitivity Q 10 g factor was different for each type of neuron .', 'This is not surprising , as it should depend on the type of channel that predominates in each neuronal type .', 'The evolution of the Q values are shown in Fig 3B for these parameters .', 'The predominant conductance IA in HVCRA is bigger compared to its Ih and the contrary happens for HVCX , so relative values coincide with conductance measurements made in the crab [49] , where values for the IA conductance are lower than 2 and near to 3 for the Ih .', 'Because our modeling does not discriminate between individual Q 10 g , we expected the relative incidence of these two conductances to dominate above the other less representative .', 'The value of 2 . 5 for HVCINT is similar to the in vivo relative current values of HVCX .', 'The final selection of parameters was assessed by finding the closest fit between modeled and experimental traces at in vivo temperatures ( See Fig 3C ) .', 'Not trivially , yet not surprisingly as was expected from the recently described dynamics of Ca2+-dependent K+ current ( ISK ) in HVCRA and HVCX , we found bursting behavior at normal bird temperatures of 40°C for these two neuronal types .', 'We used external applied currents Iapp of durations close to 10ms , which is the typical burst duration measured in vivo .', 'Once the temperature
['Introduction', 'Materials and methods', 'Results', 'Discussion']
['Different neuronal types within brain motor areas contribute to the generation of complex motor behaviors .', 'A widely studied songbird forebrain nucleus ( HVC ) has been recognized as fundamental in shaping the precise timing characteristics of birdsong .', 'This is based , among other evidence , on the stretching and the “breaking” of song structure when HVC is cooled .', 'However , little is known about the temperature effects that take place in its neurons .', 'To address this , we investigated the dynamics of HVC both experimentally and computationally .', 'We developed a technique where simultaneous electrophysiological recordings were performed during temperature manipulation of HVC .', 'We recorded spontaneous activity and found three effects: widening of the spike shape , decrease of the firing rate and change in the interspike interval distribution .', 'All these effects could be explained with a detailed conductance based model of all the neurons present in HVC .', 'Temperature dependence of the ionic channel time constants explained the first effect , while the second was based in the changes of the maximal conductance using single synaptic excitatory inputs .', 'The last phenomenon , only emerged after introducing a more realistic synaptic input to the inhibitory interneurons .', 'Two timescales were present in the interspike distributions .', 'The behavior of one timescale was reproduced with different input balances received form the excitatory neurons , whereas the other , which disappears with cooling , could not be found assuming poissonian synaptic inputs .', 'Furthermore , the computational model shows that the bursting of the excitatory neurons arises naturally at normal brain temperature and that they have an intrinsic delay at low temperatures .', 'The same effect occurs at single synapses , which may explain song stretching .', 'These findings shed light on the temperature dependence of neuronal dynamics and present a comprehensive framework to study neuronal connectivity .', 'This study , which is based on intrinsic neuronal characteristics , may help to understand emergent behavioral changes .']
['The study of the neuronal mechanisms that give rise to the complex behavior of singing in birds has been hotly debated lately .', 'Many models have been tested and novel tools have been developed to try to understand the role of a key brain nucleus in the song pathway: HVC .', 'It is believed that it is highly responsible for generating the precise timing of songs , and this has been tested by manipulating it with temperature .', 'Results showed that cooling can stretch , but that it can also restructure or “break” the song syllables .', 'However , single neuronal mechanisms are not yet described .', 'To better understand this , we cooled HVC in canaries and measured spontaneous activity electrophysiologically .', 'We found three effects: spike shape widening , spike rate reduction and changes in inter-spike-interval ( ISI ) distributions .', 'To explain them , we built a computational model with a detailed description of ion channel conductances and temperature dependency .', 'We could explain the first effect with a single neuron model .', 'The second , could be explained adding single synapses .', 'Finally , we showed similar ISI modifications of one of the timescales present by means of multiple stochastic inputs .', 'In addition , we found that excitatory neurons show natural bursting behavior at normal brain temperatures and that synaptic delays are the main candidates to explain song stretching at low temperatures .']
['action potentials', 'medicine and health sciences', 'ecology and environmental sciences', 'nervous system', 'membrane potential', 'vertebrates', 'electrophysiology', 'neuroscience', 'animals', 'physiological parameters', 'body temperature', 'computational neuroscience', 'interneurons', 'ecological metrics', 'birds', 'animal cells', 'cellular neuroscience', 'cell biology', 'ecology', 'anatomy', 'synapses', 'physiology', 'neurons', 'q10 temperature coefficient', 'single neuron function', 'biology and life sciences', 'cellular types', 'canaries', 'computational biology', 'amniotes', 'neurophysiology', 'organisms']