Simultaneous Collection of In Vivo Functional and Anatomical Data from Individual Neurons in Awake Mice

Abstract

Ideally, to trace neural circuits, one often desires access to functional data that may be linked to anatomical attributes such as neuron type or projection target. Here we describe methods used for this purpose in our laboratory. We aim for this chapter to serve as a practical guide to applying “loose patch” or “cell-attached patch” electrophysiology techniques in vivo to simultaneously obtain information about neuronal firing (e.g., responses to sensory stimuli) and detailed anatomical information including dendritic morphology and axonal targeting. Since data on neuronal circuit function are often most useful when gathered during wakeful behavior, we pay special attention here to the use of these techniques in awake, head-fixed mice. However, the same methods may easily be applied to anesthetized animals.

Appendix: Arduino Code

#include<digitalWriteFast.h> //include this library available from //https://code.google.com/p/digitalwritefast/

// Define Arduino inputs

#define c_EncoderInterrupt 0

#define c_EncoderPinA 2

#define c_EncoderPinB 4

//Define Arduino serial outputs

#define LOAD 7

#define CLOCK 8

#define DATA 9

#define LDAC 10

//Define variables

volatile bool _EncoderBSet;

volatile long _EncoderTicks = 0;

volatile long tmpdata = 0;

volatile long velocity = 0;

//set clock timing

#define HALF_CLOCK_PERIOD 10

//initialization routine

void setup()

{

//Assign functions to inputs

pinMode(c_EncoderPinA, INPUT);

digitalWrite(c_EncoderPinA, LOW);

pinMode(c_EncoderPinB, INPUT);

digitalWrite(c_EncoderPinB, LOW);

attachInterrupt(c_EncoderInterrupt, HandleMotorInterruptA, RISING);

//Assign functions to outputs

pinMode(DATA, OUTPUT);

pinMode(CLOCK,OUTPUT);

pinMode(LOAD,OUTPUT);

pinMode(LDAC,OUTPUT);

//initialize serial outputs

digitalWriteFast(DATA,LOW);

digitalWriteFast(CLOCK,HIGH);

digitalWriteFast(LOAD,HIGH);

digitalWriteFast(LDAC,HIGH);

Serial.begin (9600);

}

//Running loop function

void loop()

{

tmpdata = _EncoderTicks;

delay(40);//bin size in ms. if changed you need to change denominator in //next line

velocity = ((((_EncoderTicks-tmpdata))/0.04)*0.047) + 128; //0.047 scaling //term assumes a 6" diameter wheel

writeValue(velocity);

Serial.print(velocity-128);

Serial.print("\n");

}

// Interrupt function triggered by a click on encoder OutA

void HandleMotorInterruptA()

{

// Reading the state of encoder OutB determines the direction of rotation

if (digitalReadFast(c_EncoderPinB) == LOW) {

_EncoderTicks = _EncoderTicks-1;

} else {

_EncoderTicks = _EncoderTicks + 1;

}

}

//write value to serial connection to MAX500

void writeValue(uint8_t value)

{

//start of sequence

digitalWriteFast(DATA,LOW);

delayMicroseconds(HALF_CLOCK_PERIOD);

digitalWriteFast(CLOCK,LOW);

delayMicroseconds(HALF_CLOCK_PERIOD);

digitalWriteFast(CLOCK,HIGH);

digitalWriteFast(DATA,LOW);

delayMicroseconds(HALF_CLOCK_PERIOD);

digitalWriteFast(CLOCK,LOW);

delayMicroseconds(HALF_CLOCK_PERIOD);

digitalWriteFast(CLOCK,HIGH);

//send the 8 bit sample data

for(int i = 7;i > =0;i--){

digitalWriteFast(DATA,((value&(1 < <i)))> > i);

delayMicroseconds(HALF_CLOCK_PERIOD);

digitalWriteFast(CLOCK,LOW);

delayMicroseconds(HALF_CLOCK_PERIOD);

digitalWriteFast(CLOCK,HIGH);

}

//latch enable, DAC output is set

digitalWriteFast(DATA,LOW);

delayMicroseconds(HALF_CLOCK_PERIOD);

digitalWriteFast(LOAD,LOW);

delayMicroseconds(HALF_CLOCK_PERIOD);

delayMicroseconds(HALF_CLOCK_PERIOD);

digitalWriteFast(LOAD,HIGH);

delayMicroseconds(HALF_CLOCK_PERIOD);

digitalWriteFast(LDAC,LOW);

delayMicroseconds(HALF_CLOCK_PERIOD);

delayMicroseconds(HALF_CLOCK_PERIOD);

digitalWriteFast(LDAC,HIGH);

}