James H. Pirch

A role for acetylcholine in conditioning-related responses of rat frontal cortex neurons: microiontophoretic evidence

In an associative conditioning paradigm, an auditory stimulus (CS+) was paired with rewarding medial forebrain bundle stimulation or a tone of different frequency (CS-) was presented without pairing. After training, slow potential (SP) and single neuron responses were recorded from rat frontal cortex. When cortical SP responses indicated the development of discrimination between CS+ and CS- tones, single neurons could be isolated that exhibited a discriminative response to CS+. Seventy-three percent of the 56 neurons which discriminated between CS+ and CS- were excited by the paired tone while the remainder were inhibited. Iontophoretically applied acetylcholine increased spontaneous firing rate in 90% of the excited cells and 87% of the inhibited cells. Iontophoretic administration of a muscarinic receptor antagonist, either atropine or tropicamide, during trial presentation attenuated the conditioning-related response to CS+ as well as the response to acetylcholine in the majority of neurons. The largest group of discriminating neurons were excited by both CS+ and acetylcholine, and both responses were suppressed by the antagonists. The results provide evidence that conditioning-related responses of a major population of frontal cortex neurons are modulated by cholinergic input, a portion of which may originate in the basal forebrain area. There also may be a significant non-cholinergic influence on these neuronal responses.

Microinjection of procaine or GABA into the nucleus basalis magnocellularis affects cue-elicited unit responses in the rat frontal cortex.

Male rats were chronically implanted for recording of single units in the frontal cortex during a cue-event paradigm. The rats were sedated and restrained during the experiments. Units were selected which had large-amplitude, clearly isolated action potentials. The animals were first trained to associated a 2-s tone cue with rewarding medial forebrain bundle stimulation. After training, units responded to the cue by an increase or decrease in discharge rate. Cumulative histograms of the unit response to the cue were obtained and then either procaine hydrochloride or GABA was microinjected into the nucleus basalis magnocellularis (nBM). Immediately after drug administration another histogram was obtained to ascertain the drug effect. Procaine microinjections to the nBM suppressed the frontal cortex unit responses in 9 of 10 units that had previously responded with an increase in firing rate and 10 of 12 units that had decreased their firing rate before drug administration. GABA microinjections antagonized the response in 15 of 19 excited units and 2 of 2 inhibited units. Recovery was obtained in 23 units. Other units did not remain isolated long enough to obtain complete recovery. The nBM supplies the frontal cortex with as much as 70% of its cholinergic innervation. Lesions of the region do not significantly alter the amounts of neurotransmitters other than acetylcholine in the frontal cortex. These results indicate that neurons in the nucleus basalis magnocellularis are involved in the cue-elicited changes in the rate of discharge of units in the rat frontal cortex.

Generation of cortical event-related slow potentials in the rat involves nucleus basalis cholinergic innervation

These experiments were conducted to gather information regarding the role of cholinergic innervation to the cortex in the generation of event-related slow potentials. The effects of unilateral drug treatments or lesions on ipsilateral and contralateral frontal cortex slow potential (SP) responses were examined in rats. The SP responses were recorded with silver-silver chloride electrodes and were generated by a 2 sec light cue which preceded rewarding medial forebrain bundle stimulation. The following approaches were used: microinjection of GABA, procaine or saline into the nucleus basalis magnocellularis; microinjection of atropine or saline subdurally in the SP recording area; electrolytic lesion of the nucleus basalis area; and kainic acid lesion of the nucleus basalis area. The following bilateral measurements were obtained lesion studies: choline acetyltransferase (ChAT) in cortex and hippocampus; serotonin in cortex, hippocampus, striatum and nucleus accumbens; norepinephrine in cortex and hippocampus; dopamine in striatum and nucleus accumbens; and metabolites of serotonin, norepinephrine and dopamine in these areas. The cortical SP responses were reduced on the side ipsilateral to the injections of GABA and procaine into the nucleus basalis, and on the side of the subdural atropine injection. With either type of lesion, the SP responses on the lesioned side were significantly reduced as compared to the non-lesioned side. Reductions in cortical ChAT and other measures were observed ipsilateral to the electrolytic lesion, but only cortical ChAT activity was reduced in the kainic acid-lesioned animals. Thus, pharmacological depression of nucleus basalis neurons, blockade of cholinergic muscarinic receptors in the cortex, and nucleus basalis lesions that reduce cortical choline acetyltransferase activity depress event-related slow potentials in the rat frontal cortex. These results provide evidence that cortical slow potential responses in the rat are dependent upon cholinergic innervation from the nucleus basalis.

Basal forebrain and frontal cortex neuron responses during visual discrimination in the rat

Using a classical conditioning procedure in urethane-anesthetized rats, a light applied to one eye (CS+) was paired with medial forebrain bundle (MFB) stimulation, whereas a light applied to the other eye (CS-) was not paired. Basal forebrain neurons in the substantia innominata, medial globus pallidus, and nucleus basalis magnocellularis responded differentially to CS+ and CS-, with larger responses to CS+. Some neurons were excited by CS+, and others were inhibited. Fifty percent of these neurons responded in the same direction to CS+ and MFB stimulation, and 38% responded in opposite directions. Frontal cortex neurons exhibited similar differential responses; 47% of the differential responses to CS+ were in the same direction as the response to MFB stimulation, and 29% were in the opposite direction. When light to either eye was paired with MFB stimulation, conditioning-related basal forebrain neuron responses of comparable magnitude to left and right eye illumination were observed, providing evidence that association of CS and UCS rather than the eye to which light was applied determined the differential response to CS+. Also, two different intensities of light induced comparable basal forebrain responses when both were paired with the UCS. These experiments provide support for a role of the basal forebrain in conditioning-related neural activity. Furthermore, this preparation can be utilized to investigate transmitter systems that mediate conditioning-related responses of basal forebrain neurons.

Event-Related Slow Potentials and Activity of Single Neurons in Rat Frontal Cortex

Event-related slow potentials and single unit activity were recorded from frontal cortex of unanesthetized, restrained rats trained to associate an auditory cue with rewarding medial forebrain bundle (MFB) stimulation. The majority of cortical units demonstrated an alteration of firing rate after the cue, with the predominant response being excitatory. The patterns of unit response during the interval between the cue and reinforcement varied considerable and few units showed a pattern which coincided temporally with the wave forms of the negative slow potential responses. These results indicate that negative cortical slow potential responses to meaningful stimuli are associated with an overall increase in activity of cortical neurons.

Single-unit and slow potential responses from rat frontal cortex during associative conditioning.

Single-unit and slow potential responses were recorded from the frontal cortex of unanesthetized, restrained rats trained to associate an auditory cue (tone) with rewarding medial forebrain bundle stimulation. Slow potential responses to the unpaired tone were minimal whereas large negative slow potential responses developed to the paired tone. Units were selected which had large-amplitude action potentials and positive first deflections, characteristics suggesting that the recordings were derived from pyramidal neurons. Responses of excitation, inhibition, and no change were observed during tone presentation. Forty-five percent of the units responded to the unpaired tone; with pairing, 90% of the units demonstrated significant responses. Furthermore, the magnitude of the excitatory responses was enhanced by pairing and a distribution pattern developed in which the overall response of the more superficial units was activation whereas deeper units were inhibited. The results suggest that the conditioning-related negative slow potential responses recorded from the surface of the rat frontal cortex reflect excitatory processes which are associated with an enhanced firing rate of neurons in the upper layers.

Basal Forebrain Modulation of Cortical Cell Activity During Conditioning

Several investigators have found that basal forebrain neurons in the area of the nucleus basalis and substantia innominata respond to cues that signal the availability of reinforcement (DeLong, 1971; Mitchell et al., 1987; Richardson and DeLong, 1986, 1988; Rolls et al., 1979; Travis and Sparks, 1968; Wilson and Rolls, 1990a, 1990b). The reinforcement-related or conditioning-related responses of such basal forebrain neurons do not depend upon the sensory modality of the signal cue (Wilson and Rolls, 1990a). It is also known that cortical neurons respond to conditioned stimuli applied during similar behavioral paradigms (Aou et al., 1983; Boyd et al., 1982; Fuster et al., 1982; Kojima and Goldman-Rakic, 1982; Mauritz and Wise, 1986; Peterson, 1986; Watanabe, 1990), suggesting a possible relationship between cortical neuron responses and activity of basal forebrain neurons.

Amphetamine effects on brain slow potentials associated with discrimination in the rat.

Slow potentials were recorded from the anterior cortex of rats during discrimination conditioning and the effects of various doses of d-amphetamine on these responses were examined. In the discrimination paradigm one tone (Sd) was followed at three see after the onset by food reinforcement while another tone (S delta) indicated that no reinforcement would follow. Slow potential (SP) responses were measured during the three-sec period following onset of the stimulus. For the first several training sessions the SP responses demonstrated a phase of generalization during which responses were the same to both stimuli. Thereafter, the responses to Sd were significantly greater than responses to S delta. d-Amphetamine produced a dose-related depression of SP responses to the reinforced stimulus in doses of 0.25 to 2.0 mg/kg. The effect of amphetamine on SP responses to S delta was biphasic; the lower doses (0.25 and 0.5 mg/kg) enhanced responses, no change was seen after 1.0 mg/kg and the high dose (2.0 mg/kg) depressed responses. This study demonstrates that the rat develops differential slow potential responses to reinforced and nonreinforced stimuli in a discrimination paradigm and that d-amphetamine produces a differential and dose-related alteration of these SP responses. It is suggested that the actions of amphetamine may be produced through interference with mechanisms of discrimination, by an effect on subcortical activating systems involving norepinephrine, and/or by activation of inhibitory dopamine receptors on cortical neurons.

Conditioning-Related Single Unit Activity in the Frontal Cortex of Urethane Anesthetized Rats

Responses of frontal cortex single units to a tone preceding medial forebrain bundle (MFB) stimulation were recorded in urethane anesthetized rats. The animals were implanted with monopolar electrodes for MFB stimulation and, following recovery, stimulation parameters which supported self-stimulation were determined for each rat. Prior to the unit recording experiment, the animals were trained to associate a 2-sec tone with MFB stimulation. Trials were presented at variable intervals. Under urethane anesthesia, single units were isolated and the responses of units to paired and unpaired tones were determined. The results indicate that conditioning-related responses of frontal cortex single units can be recorded in urethane anesthetized rats.

Conditioned Cortical Slow Potential Responses in Urethane Anesthetized Rats

Cortical slow potential (SP) responses to tone or light stimuli preceding medial forebrain bundle (MFB) stimulation were recorded in urethane anesthetized rats. In the first study, rats were implanted with Ag-AgCl electrodes for recording frontal cortex SPs as well as monopolar electrodes for MFB stimulation. Following recovery, optimum stimulation parameters for SP conditioning were determined for each rat during self-stimulation sessions. These animals were then subjected to extensive associative conditioning in the unanesthetized state. Trials were presented at variable intervals and a 2-sec tone preceded a single 0.5 sec train of MFB stimulation. Negative SP responses developed with training and responses of similar waveform and amplitude were observed in the same animals under urethane anesthesia. Other rats were implanted with MFB stimulating electrodes and, after recovery, stimulation parameters were determined as above but the animals were not subjected to the conditioning procedure prior to urethane administration. Under urethane anesthesia, Ag-AgCl electrodes were placed on the dura over frontal cortex for recording SP responses during pseudoconditioning, conditioning, extinction and retraining trials, using either light or tone stimuli. Negative bilateral SP responses to the tone or light were minimal or nonexistent during pseudoconditioning, developed gradually with pairing, diminished markedly during extinction and returned to maximum amplitude with retraining. The SP responses also reflected discrimination between reinforced and nonreinforced tone and light stimuli as well as reversal conditioning. Furthermore, turning off a light could also serve as the conditioned stimulus for SP response generation. Cortical slow potential responses can be conditioned in urethane anesthetized rats. Therefore, it may be possible to apply additional neurophysiological techniques in these animals to investigate event-related slow potential mechanisms.