Transgenic silencing of neurons in the mammalian brain by expression of the allatostatin receptor (AlstR).

The mammalian brain is an enormously complex set of circuits composed of interconnected neuronal cell types. The analysis of central neural circuits will be greatly served by the ability to turn off specific neuronal cell types while recording from others in intact brains. Because drug delivery cannot be restricted to specific cell types, this can only be achieved by putting "silencer" transgenes under the control of neuron-specific promoters. Towards this end we have created a line of transgenic mice putting the Drosophila allatostatin (AL) neuropeptide receptor (AlstR) under the control of the tetO element, thus enabling its inducible expression when crossed to tet-transactivator lines. Mammals have no endogenous AL or AlstR, but activation of exogenously expressed AlstR in mammalian neurons leads to membrane hyperpolarization via endogenous G-protein-coupled inward rectifier K(+) channels, making the neurons much less likely to fire action potentials. Here we show that this tetO/AlstR line is capable of broadly expressing AlstR mRNA in principal neurons throughout the forebrain when crossed to a commercially-available transactivator line. We electrophysiologically characterize this cross in hippocampal slices, demonstrating that bath application of AL leads to hyperpolarization of CA1 pyramidal neurons, making them refractory to the induction of action potentials by injected current. Finally, we demonstrate the ability of AL application to silence the sound-evoked spiking responses of auditory cortical neurons in intact brains of AlstR/tetO transgenic mice. When crossed to other transactivator lines expressing in defined neuronal cell types, this AlstR/tetO line should prove a very useful tool for the analysis of intact central neural circuits.

Connections of the caudal anterior cingulate cortex in rabbit: neural circuitry participating in the acquisition of trace eyeblink conditioning.

The caudal anterior cingulate cortex (cAC) is an essential component of the circuitry involved in acquisition of forebrain-dependent trace eyeblink conditioning. Lesions of the cAC prevent trace eyeblink conditioning [Weible AP, McEchron MD, Disterhoft JF (2000) Cortical involvement in acquisition and extinction of trace eyeblink conditioning. Behav Neurosci 114(6):1058-1067]. The patterns of activation of cAC neurons recorded in vivo suggest an attentional role for this structure early in training [Weible AP, Weiss C, Disterhoft JF (2003) Activity profiles of single neurons in caudal anterior cingulate cortex during trace eyeblink conditioning in the rabbit. J Neurophysiol 90(2):599-612]. The goal of the present study was to identify connections of the portion of the rabbit cAC previously demonstrated to be involved in trace eyeblink conditioning, using the neuronal tract tracer wheat germ agglutinin conjugated to horseradish peroxidase, to better understand how the cAC contributes to the process of associative learning. Reciprocal connections with the claustrum provide a route for the transfer of sensory information between the cAC and neocortical and allocortical regions also involved in learning. Connections with components of the basal forebrain cholinergic system are described, with relevance to the proposed attentional role of the cAC. Reciprocal and unidirectional connections were in evidence in multiple thalamic regions, including the medial dorsal nucleus, which have been implicated in a variety of conditioning paradigms. Anterograde connections with the caudate and lateral pontine nuclei provide access to forebrain motor and brainstem sensory circuitry, respectively. The relevance of these connections to acquisition of the trace conditioned reflex is discussed.

Comparisons of dorsal and ventral hippocampus cornu ammonis region 1 pyramidal neuron activity during trace eye-blink conditioning in the rabbit.

Previous studies demonstrating a critical role of the hippocampus during trace eye-blink conditioning have focused primarily upon the dorsal portion of the structure. However, evidence suggests that a functional differentiation exists along the septotemporal axis of the hippocampus. In the present study, the activity of 2588 single cornu ammonis region 1 pyramidal neurons of the dorsal hippocampus and ventral hippocampus were recorded during trace and pseudo-eye-blink conditioning of the rabbit. Learning-related increases in dorsal hippocampus neuron firing rates were observed immediately prior to behavioral criterion, and increased over the course of training. Activation of dorsal hippocampus neurons during trace conditioning was also greater than that of ventral hippocampus neurons, including during the trace interval, in well-trained animals. An unexpected difference in the patterns of learning-related activity between hemispheres was also observed. Neurons of the dorsal hippocampus ipsilateral and contralateral to the trained eye, exhibiting significant increases in firing rate [rate increasing neurons], demonstrated the greatest magnitude of activation early and late in training, respectively. Rate increasing neurons of the dorsal hippocampus also exhibited a greater diversity of response profiles, with 69% of dorsal hippocampus rate increasing neurons exhibiting significant increases in firing rate during the conditioned stimulus and/or trace intervals, compared with only 8% of ventral hippocampus rate increasing neurons (the remainder of which were significantly responsive during only the unconditioned stimulus and/or post-unconditioned stimulus intervals). Only modest learning-related activation of ventral hippocampus neurons was observed, reflected as an increase in conditioning stimulus-elicited rate increasing neuron response magnitudes over the course of training. No differences in firing rate between dorsal hippocampus and ventral hippocampus neurons during a 1-day pre-training habituation session were observed. Thus, dorsal hippocampus activation is more robust, suggesting a more substantial role for these neurons in the processing of temporal information during trace eye-blink conditioning.

Aging and learning-specific changes in single-neuron activity in CA1 hippocampus during rabbit trace eyeblink conditioning.

Rabbit trace eyeblink conditioning is a hippocampus-dependent task in which the auditory conditioned stimulus (CS) is separated from the corneal airpuff unconditioned stimulus (US) by a 500-ms empty trace interval. Young rabbits are able to associate the CS and US and acquire trace eyeblink conditioned responses (CRs); however, a subset of aged rabbits show poor learning on this task. Several studies have shown that CA1-hippocampal activity is altered by aging; however, it is unknown how aging affects the interaction of CA1 single neurons within local ensembles during learning. The present study examined the extracellular activity of CA1 pyramidal neurons within local ensembles in aged (29-34 mo) and young (3-6 mo) rabbits during 10 daily sessions (80 trials/session) of trace eyeblink conditioning. A single surgically implanted nonmovable stereotrode was used to record ensembles ranging in size from 2 to 12 separated single neurons. A total of six young and four aged rabbits acquired significant levels of CRs, whereas five aged rabbits showed very few CRs similar to a group of five young pseudoconditioned rabbits. Pyramidal cells (2,159 total) were recorded from these four groups during training. Increases in CA1 pyramidal cell firing to the CS and US were diminished in the aged nonlearners. Local ensembles from all groups contained heterogeneous types of pyramidal cell responses. Some cells showed increases while others showed decreases in firing during the trace eyeblink trial. Hierarchical clustering was used to isolate seven different classes of single-neuron responses that showed unique firing patterns during the trace conditioning trial. The proportion of cells in each group was similar for six of seven response classes. Unlike the excitatory modeling patterns reported in previous studies, three of seven response types (67% of recorded cells) exhibited some type of inhibitory decrease to the CS, US, or both. The single-neuron response classes showed different patterns of learning-related activity across training. Several of the single-neuron types from the aged nonlearners showed unique alterations in response magnitude to the CS and US. Cross-correlation analyses suggest that specific single-neuron types provide more correlated single-neuron activity to the ensemble processing of information. However, aged nonlearners showed a significantly lower level of coincident pyramidal cell firing for all cell types within local ensembles in CA1.

Cortical involvement in acquisition and extinction of trace eyeblink conditioning.

Previous studies have implicated 2 cortical regions interconnected with the hippocampal formation, the retrosplenial cortex (RSC) and the medial prefrontal cortex (mPFC), as loci important for the acquisition of hippocampally dependent trace eyeblink conditioning. These loci have also been proposed to serve as long-term storage sites of task critical information. This study used lesions made prior to training to investigate the roles of the RSC, as well as the caudal and rostral subdivisions of the mPFC, in the acquisition and subsequent extinction of trace eyeblink conditioning in the rabbit. The caudal mPFC and rostral mPFC were shown to be critical for acquisition and extinction of the conditioned reflex, respectively. The data indicate that the RSC is not critical for acquisition or extinction of the trace conditioned reflex.