Todor V. Gerdjikov

A major external source of cholinergic innervation of the striatum and nucleus accumbens originates in the brainstem

Cholinergic transmission in the striatal complex is critical for the modulation of the activity of local microcircuits and dopamine release. Release of acetylcholine has been considered to originate exclusively from a subtype of striatal interneuron that provides widespread innervation of the striatum. Cholinergic neurons of the pedunculopontine (PPN) and laterodorsal tegmental (LDT) nuclei indirectly influence the activity of the dorsal striatum and nucleus accumbens through their innervation of dopamine and thalamic neurons, which in turn converge at the same striatal levels. Here we show that cholinergic neurons in the brainstem also provide a direct innervation of the striatal complex. By the expression of fluorescent proteins in choline acetyltransferase (ChAT)::Cre+ transgenic rats, we selectively labeled cholinergic neurons in the rostral PPN, caudal PPN, and LDT. We show that cholinergic neurons topographically innervate wide areas of the striatal complex: rostral PPN preferentially innervates the dorsolateral striatum, and LDT preferentially innervates the medial striatum and nucleus accumbens core in which they principally form asymmetric synapses. Retrograde labeling combined with immunohistochemistry in wild-type rats confirmed the topography and cholinergic nature of the projection. Furthermore, transynaptic gene activation and conventional double retrograde labeling suggest that LDT neurons that innervate the nucleus accumbens also send collaterals to the thalamus and the dopaminergic midbrain, thus providing both direct and indirect projections, to the striatal complex. The differential activity of cholinergic interneurons and cholinergic neurons of the brainstem during reward-related paradigms suggest that the two systems play different but complementary roles in the processing of information in the striatum.

Place preference induced by nucleus accumbens amphetamine is impaired by antagonists of ERK or p38 MAP kinases in rats.

The nucleus accumbens (NAc) plays a role in conditioned place preference (CPP). The authors tested the hypothesis that inhibition of mitogen-activated protein kinases (MAPKs) would inhibit NAc-amphetamine-produced CPP. Results confirmed that NAc amphetamine increased levels of the MAPK extracellular signal-regulated kinase (ERK). In CPP studies, NAc injections (0.5 microl per side) of the ERK inhibitor PD98059 (1.0-2.5 microg) or the p38 kinase inhibitor SB203580 (15-500 ng) dose dependently impaired CPP. The c-Jun-N-terminal kinase (JNK) inhibitor SP600125 (1.0-2.5 microg) failed to block the CPP effect. The drugs did not block amphetamine-induced motor activity. Results suggest that ERK and p38, but not JNK, MAPKs may be necessary for the establishment of NAc amphetamine-produced CPP and may also mediate other forms of reward-related learning dependent on NAc.

The head-fixed behaving rat--procedures and pitfalls.

This paper describes experimental techniques with head-fixed, operantly conditioned rodents that allow the control of stimulus presentation and tracking of motor output at hitherto unprecedented levels of spatio-temporal precision. Experimental procedures for the surgery and behavioral training are presented. We place particular emphasis on potential pitfalls using these procedures in order to assist investigators who intend to engage in this type of experiment. We argue that head-fixed rodent models, by allowing the combination of methodologies from molecular manipulations, intracellular electrophysiology, and imaging to behavioral measurements, will be instrumental in combining insights into the functional neuronal organization at different levels of observation. Provided viable behavioral methods are implemented, model systems based on rodents will be complementary to current primate models—the latter providing highest comparability with the human brain, while the former offer hugely advanced methodologies on the lower levels of organization, for example, genetic alterations, intracellular electrophysiology, and imaging.

The Role of Signaling Molecules in Reward-Related Incentive Learning

Reward-related incentive learning involves the acquisition by neutral stimuli of an enhanced ability to elicit approach and other responses. Previous studies have shown that both dopamine (DA) and glutamate (Glu) play critical roles in this type of learning. Signaling molecules are intracellular messengers that participate in the influence of transmitter-receptor events on intracellular function including transcription in the nucleus. In recent years studies have begun to implicate signaling molecules in incentive learning. Thus, inhibition of cyclic adenosine monophosphate-dependent protein kinase (PKA) in the nucleus accumbens (NAc), that is activated by DA acting at D1-like receptors, blocks the acquisition of conditioned approach responses, lever pressing for food, conditioned place preference (CPP) based on NAc injections of amphetamine or cocaine, and conditioned activity based on NAc injections of amphetamine. Similar effects have been observed with PKA inhibition in the basolateral amygdala or medial prefrontal cortex. If animals were trained prior to testing with PKA inhibitors in NAc, no effect was seen suggesting that PKAis more important for acquisition than expression of incentive learning.Inhibition of calcium-dependent protein kinase or mitogen-activated protein kinases in NAc similarly has been shown to block the acquisition of incentive learning. Results support a model of DA-Glu synaptic interactions that form the basis of incentive learning.

Segregated cholinergic transmission modulates dopamine neurons integrated in distinct functional circuits.

Dopamine neurons in the ventral tegmental area (VTA) receive cholinergic innervation from brainstem structures that are associated with either movement or reward. Whereas cholinergic neurons of the pedunculopontine nucleus (PPN) carry an associative/motor signal, those of the laterodorsal tegmental nucleus (LDT) convey limbic information. We used optogenetics and in vivo juxtacellular recording and labeling to examine the influence of brainstem cholinergic innervation of distinct neuronal subpopulations in the VTA. We found that LDT cholinergic axons selectively enhanced the bursting activity of mesolimbic dopamine neurons that were excited by aversive stimulation. In contrast, PPN cholinergic axons activated and changed the discharge properties of VTA neurons that were integrated in distinct functional circuits and were inhibited by aversive stimulation. Although both structures conveyed a reinforcing signal, they had opposite roles in locomotion. Our results demonstrate that two modes of cholinergic transmission operate in the VTA and segregate the neurons involved in different reward circuits.

Hippocampal α5 subunit-containing GABAA receptors are involved in the development of the latent inhibition effect

Hippocampal GABA(A) receptors containing the alpha 5 subunit have been implicated in the modulation of hippocampal-dependent learning, presumably via their tonic inhibitory influence on hippocampal glutamatergic activity. Here, we examined the expression of latent inhibition (LI)--a form of selective learning that is sensitive to a number of manipulations targeted at the hippocampal formation, in alpha 5(H105R) mutant mice with reduced levels of hippocampal alpha 5-containing GABA(A) receptors. A single pre-exposure to the taste conditioned stimulus (CS) prior to the pairing of the same CS with LiCl-induced nausea was effective in reducing the conditioned aversion against the taste CS in wild-type mice--thus constituting the LI effect. LI was however distinctly absent in male alpha 5(H105R) mutant mice. Hence, a partial loss of hippocampal alpha 5 GABA(A) receptors is sufficient to alter one major form of selective learning, albeit this was not seen in the female. This observed phenotype suggests that specific activation of these extrasynaptic GABA(A) receptors may confer therapeutic potential against the failure to show selectivity in learning by human psychotic patients.

Extrinsic Sources of Cholinergic Innervation of the Striatal Complex: A Whole-Brain Mapping Analysis

Acetylcholine in the striatal complex plays an important role in normal behavior and is affected in a number of neurological disorders. Although early studies suggested that acetylcholine in the striatum (STR) is derived almost exclusively from cholinergic interneurons (CIN), recent axonal mapping studies using conditional anterograde tracing have revealed the existence of a prominent direct cholinergic pathway from the pedunculopontine and laterodorsal tegmental nuclei to the dorsal striatum and nucleus accumbens. The identification of the importance of this pathway is essential for creating a complete model of cholinergic modulation in the striatum, and it opens the question as to whether other populations of cholinergic neurons may also contribute to such modulation. Here, using novel viral tracing technologies based on phenotype-specific fluorescent reporter expression in combination with retrograde tracing, we aimed to define other sources of cholinergic innervation of the striatum. Systematic mapping of the projections of all cholinergic structures in the brain (Ch1 to Ch8) by means of conditional tracing of cholinergic axons, revealed that the only extrinsic source of cholinergic innervation arises in the brainstem pedunculopontine and laterodorsal tegmental nuclei. Our results thus place the pedunculopontine and laterodorsal nuclei in a key and exclusive position to provide extrinsic cholinergic modulation of the activity of the striatal systems.

Differential effects of calcineurin inhibition and protein kinase A activation on nucleus accumbens amphetamine-produced conditioned place preference in rats.

The nucleus accumbens (NAc) plays a critical role in amphetamine‐produced conditioned place preference (CPP). In previous studies inhibition or activation of cyclic adenosine monophosphate‐dependent protein kinase (PKA) blocked NAc amphetamine‐produced CPP. PKA activation unrelated to ongoing DA transmission may disrupt reward‐related learning. Calcineurin (CN) down‐regulates downstream PKA targets. Unlike PKA activation, CN inhibition may preserve and enhance reward‐related learning. The PKA signalling cascade is negatively regulated by calcineurin (CN). We tested the hypothesis that post‐training CN inhibition in NAc will enhance NAc amphetamine‐produced CPP and that PKA activation will block CPP. Eight but not four or two 30‐min conditioning sessions were sufficient to establish significant CPP. Immediate post‐training, NAc injection of the calcineurin inhibitor FK506 (5.0 but not 1.0 µg in 0.5 µL per side) led to a significant amphetamine CPP in rats receiving four but not two training sessions; the 5.0‐µg dose had no effect on rats trained with eight sessions. Injections of the PKA activator Sp‐cAMPS (2.5 or 10.0 µg in 0.5 µL per side) failed to affect CPP following two or four training sessions and blocked CPP produced by a standard 8‐day conditioning schedule. Results suggest that CN acts as a negative regulator in the establishment of NAc amphetamine‐produced CPP, a form of reward‐related learning.

Rhythmic Whisking Area (RW) in Rat Primary Motor Cortex: An Internal Monitor of Movement-Related Signals?

Vibrissae-related sensorimotor cortex controls whisking movements indirectly via modulation of lower-level sensorimotor loops and a brainstem central pattern generator (CPG). Two different whisker representations in primary motor cortex (vM1) affect whisker movements in different ways. Prolonged microstimulation in RF, a larger anterior subregion of vM1, gives rise to complex face movements and whisker retraction while the same stimulation evokes large-amplitude rhythmic whisker movement in a small caudo-medial area (RW). To characterize the motor cortex representation of explorative whisking movements, here we recorded RW units in head-fixed rats trained to contact a moving object with one whisker. RW single units were found to encode two aspects of whisker movement independently, albeit on slow time scales (hundreds of milliseconds). The first is whisker position. The second consists of speed (absolute velocity), intensity (instantaneous power), and frequency (spectral centroid). The coding for the latter three parameters was tightly correlated and realized by a continuum of RW responses—ranging from a preference of movement to a preference of rest. Information theory analysis indicated that RW spikes carry most information about position and frequency, while intensity and speed are less well represented. Further, investigating multiple and single RW units, we found a lack of phase locking, movement anticipation, and contact-related tactile responses. These findings suggest that RW neither programs detailed whisker trajectories nor initiates them. Nor does it play a role in processing object touch. Its relationship to whisking is thus indirect and may be related to movement monitoring, perhaps using feedback from the CPG.

Dopamine-Glutamate Interactions in Reward-Related Incentive Learning

Extensive evidence implicates the neurotransmitter dopamine (DA) in reward-related incentive learning (for reviews, see refs. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13). DA projections to the nucleus accumbens (NAc; refs. 14, 15, 16, 17), striatum (18), amygdala (19), and medial prefrontal cortex (mPFC; ref. (20) have been shown to be involved. In recent years, researchers have begun to focus on the neurochemical mechanisms underlying the role of DA in learning and significant advances have been made (21, 22, 23). Many data suggest that DA afferents interact with glutamatergic (Glu) afferents common to the same cell when reward-related learning occurs (see ref. 22). Results further suggest that a number of signaling molecules activated by Glu and DA synaptic transmission interact to bring about short-term and long-term alterations that mediate the neurochemical and structural changes that form the basis of reward-related incentive learning (see ref. 22). In this chapter, we will review some of the studies examining the role of DA and especially Glu neurotransmission in reward-related learning. This will be followed by a discussion of evidence that provides a basis for understanding the DA-Glu interactions and the signaling pathways that mediate the effects of reward on behavior. Finally, the role of Glu in reward-related learning will be considered from the point of view of this evidence.