Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons

The cerebral cortex is characterized by multiple layers and many distinct cell-types that together as a network are responsible for many higher cognitive ...

VU University Amsterdam

With a combined in vitro/in vivo electrophysiological and behavioral approach, we have correlated conditioned fear behavior to electrophysiological activities in the lateral amygdala and the hippocampal formation in rodents. Data indicate that projection neurons in the lateral amygdala display a continuum of spike patterns including accommodating patterns, regular firing, and oscillatory activity at theta frequencies. The firing pattern is controlled to an important part by the intracellular cAMP system, in that an increase in intracellular cAMP concentration facilitates regular firing and theta oscillations. Oscillatory electrical activity, in turn, provides an important cellular element of synchronized theta activity at 4-8 Hz (indicating atropine-sensitive type 2 theta) occurring in amygdalo-hippocampal pathways during conditioned fear responses. This type of rhythmic network activity is associated with the retrieval of long-term fear memory following cued and contextual fear conditioning, but is not related to the expression of fear behavior per se or to short-term fear memory. Synchronization at theta frequencies is suggested to represent activity in amygdalo-hippocampal pathways associated with system consolidation of fear memory, which is supported by the cholinergic system.

Theta activity in neurons and networks of the amygdala related to long-term fear memory.

After fear conditioning, plastic changes of excitatory synaptic transmission occur in the amygdala. Fear-related memory also involves the GABAergic system, although no influence on inhibitory synaptic transmission is known. In the present study we assessed the influence of Pavlovian fear conditioning on the plasticity of GABAergic synaptic interactions in the lateral amygdala (LA) in brain slices prepared from fear-conditioned, pseudo-trained and naïve adult mice. Theta-burst tetanization of thalamic afferent inputs to the LA evoked an input-specific potentiation of inhibitory postsynaptic responses in projection neurons; the cortical input was unaffected. Philanthotoxin (10 microM), an antagonist of Ca2+-permeable AMPA receptors, disabled this plastic phenomenon. Surgical isolation of the LA, extracellular application of a GABA(B) receptor antagonist (CGP 55845A, 10 microM) or an NMDA receptor antagonist (APV, 50 microM), or intracellular application of BAPTA (10 mM), did not influence the plasticity. The plasticity also showed as a potentiation of monosynaptic excitatory responses in putative GABAergic interneurons. Pavlovian fear conditioning, but not pseudo-conditioning, resulted in a significant reduction in this potentiation that was evident 24 h after training. Two weeks after training, the potentiation returned to control levels. In conclusion, a reduction in potentiation of inhibitory synaptic interactions occurs in the LA and may contribute to a shift in synaptic balance towards excitatory signal flow during the processes of fear-memory acquisition or consolidation.

Plasticity of inhibitory synaptic network interactions in the lateral amygdala upon fear conditioning in mice.

The amygdala and the hippocampus are critically involved in the formation and retention of fear memories. However, their precise contribution to, and their interplay during, fear memory formation are not fully understood. In the present study we investigated network activities in the amygdalo-hippocampal system of freely behaving mice at different stages of fear memory consolidation and retention. Our data show enhanced theta phase synchronization in this pathway during the retrieval of fear memory at long-term (24 h post-training), but not short-term (2 min, 30 min and 2 h post-training) stages, following both contextual and auditory cued conditioning. However, retrieval of remotely conditioned fear (30 days post-training) failed to induce an increase in synchronization despite there still being memory retention. Thus, our data indicate that the amygdalo-hippocampal interaction reflects a dynamic interaction of ensemble activities related to various stages of fear memory consolidation and/or retention, and support the notion that recent and remote memories are organized through different network principles.

Dissociated theta phase synchronization in amygdalo- hippocampal circuits during various stages of fear memory.

We have recently demonstrated high theta-phase synchronization between the lateral amygdala and CA1 area of the hippocampus during retrieval of long-term (1 day) fear memory, and not during short-term (2 h) or remote memory retrieval (30 days). These results indicated that the amygdalo-hippocampal interaction reflects a dynamic change of ensemble activities related to various stages of fear memory storage. In this study, we investigated theta activity during the reconsolidation of a remote contextual fear memory by re-exposing animals to the shock context 30 days after training. Consistent with our previous results, high theta synchronization was no longer apparent during re-exposure to the shock context, but was significantly higher 1 day after context re-exposure. These data indicate that the reconsolidation of remote contextual fear memory includes changes in ensemble activities between the lateral amygdala and CA1.

Theta resynchronization during reconsolidation of remote contextual fear memory.

Evidence suggests that plasticity of the amygdalar and hippocampal GABAergic system is critical for fear memory formation. In this study we investigated in wild-type and genetically manipulated mice the role of the activity-dependent 65-kDa isozyme of glutamic acid decarboxylase (GAD65) in the consolidation and generalization of conditioned fear. First, we demonstrate a transient reduction of GAD65 gene expression in the dorsal hippocampus (6 h post training) and in the basolateral complex of the amygdala (24 h post training) during distinct phases of fear memory consolidation. Second, we show that targeted ablation of the GAD65 gene in Gad65(-/-) mice results in a pronounced context-independent, intramodal generalization of auditory fear memory during long-term (24 h or 14 d) but not short-term (30 min) memory retrieval. The temporal specificity of both gene regulation and memory deficits in Gad65 mutant mice suggests that GAD65-mediated GABA synthesis is critical for the consolidation of stimulus-specific fear memory. This function appears to involve a modulation of neural activity patterns in the amygdalo-hippocampal pathway as indicated by a reduction in theta frequency synchronization between the amygdala and hippocampus of Gad65(-/-) mice during the expression of generalized fear memory.

Critical role of the 65-kDa isoform of glutamic acid decarboxylase in consolidation and generalization of Pavlovian fear memory.

Extinction procedures are clinically relevant for reducing pathological fear, and the mechanisms of fear regulation are a subject of intense research. The amygdala, hippocampus, and prefrontal cortex (PFC) have all been suggested to be key brain areas in extinction of conditioned fear. GABA has particularly been implicated in extinction learning, and the 65 kDa isoform of glutamic acid decarboxylase (GAD65) may be important in elevating GABA levels in response to environmental signals. Extinction of conditioned fear was examined in Gad65(-/-) mice while recording local field potentials from the amygdala, hippocampus, and PFC simultaneously while monitoring behavior. Gad65(-/-) mice showed generalization of cued fear, as reported previously, and impaired extinction of cued fear, such that fear remained high across extinction training. This endurance in cued fear was associated with theta frequency synchronization between the amygdala and hippocampus. Extinction of contextual fear, however, was unaltered in Gad65(-/-) mice when compared with wild-type littermates. The data imply that GAD65 plays a critical role in regulating cued fear responses during extinction learning and that, during this process, GABAergic signaling is involved in modulating synchronized activity between the amygdala and hippocampus. In view of the more pronounced effect on cued versus contextual fear extinction, these influences may rely more on GABAergic mechanisms in the amygdala.

Deficiency of the 65 kDa isoform of glutamic acid decarboxylase impairs extinction of cued but not contextual fear memory.

The relationship between epilepsy and fear has received much attention. However, seizure-modulated fear and physiologic or structural correlates have not been examined systematically, and the underlying basics of network levels remain unclear to date. Therefore, this project was set up to characterize the neurophysiologic basis of seizure-related fear and the contribution of the amygdala-hippocampus system.

Impaired extinction of fear and maintained amygdala-hippocampal theta synchrony in a mouse model of temporal lobe epilepsy.

Signals related to fear memory and extinction are processed within brain pathways involving the lateral amygdala (LA) for formation of aversive stimulus associations, the CA1 area of the hippocampus for context-dependent modulation of these associations, and the infralimbic region of the medial prefrontal cortex (mPFC) for extinction processes. While many studies have addressed the contribution of each of these modules individually, little is known about their interactions and how they function as an integrated system. Here we show, by combining multiple site local field potential (LFP) and unit recordings in freely behaving mice in a fear conditioning paradigm, that theta oscillations may provide a means for temporally and functionally connecting these modules. Theta oscillations occurred with high specificity in the CA1-LA-mPFC network. Theta coupling increased between all areas during retrieval of conditioned fear, and declined during extinction learning. During extinction recall, theta coupling partly rebounded in LA-mPFC and CA1-mPFC, and remained at a low level in CA1-LA. Interfering with theta coupling through local electrical microstimulation in CA1-LA affected conditioned fear and extinction recall depending on theta phase. These results support the hypothesis that theta coupling provides a means for inter-areal coordination in conditioned behavioral responsiveness. More specifically, theta oscillations seem to contribute to a population code indicating conditioned stimuli during recall of fear memory before and after extinction.

Patterns of coupled theta activity in amygdala-hippocampal-prefrontal cortical circuits during fear extinction.

The three-dimensional (3D) structure of neural circuits is commonly studied by reconstructing individual or small groups of neurons in separate preparations. Investigation of structural organization principles or quantification of dendritic and axonal innervation thus requires integration of many reconstructed morphologies into a common reference frame. Here we present a standardized 3D model of the rat vibrissal cortex and introduce an automated registration tool that allows for precise placement of single neuron reconstructions. We (1) developed an automated image processing pipeline to reconstruct 3D anatomical landmarks, i.e., the barrels in Layer 4, the pia and white matter surfaces and the blood vessel pattern from high-resolution images, (2) quantified these landmarks in 12 different rats, (3) generated an average 3D model of the vibrissal cortex and (4) used rigid transformations and stepwise linear scaling to register 94 neuron morphologies, reconstructed from in vivo stainings, to the standardized cortex model. We find that anatomical landmarks vary substantially across the vibrissal cortex within an individual rat. In contrast, the 3D layout of the entire vibrissal cortex remains remarkably preserved across animals. This allows for precise registration of individual neuron reconstructions with approximately 30 µm accuracy. Our approach could be used to reconstruct and standardize other anatomically defined brain areas and may ultimately lead to a precise digital reference atlas of the rat brain.

3D reconstruction and standardization of the rat vibrissal cortex for precise registration of single neuron morphology.

Cortical pyramidal neurons show irregular in vivo action potential (AP) spiking with high-frequency bursts occurring on sparse background activity. Somatic APs can backpropagate from soma into basal and apical dendrites and locally generate dendritic calcium spikes. The critical AP frequency for generation of such dendritic calcium spikes can be very different depending on cell type or brain area involved. Previously, it was shown in vitro that calcium electrogenesis can be induced in L(ayer) 5 pyramidal neurons of prefrontal cortex (PFC). It remains an open question whether somatic burst spiking and the resulting dendritic calcium electrogenesis also occur in morphologically more compact L2/3 pyramidal neurons. Furthermore, it is not known whether critical frequencies that trigger dendritic calcium electrogenesis occur in PFC under awake conditions in vivo. Here, we addressed these issues and found that pyramidal neurons in both PFC L2/3 and L5 in awake rats spike APs in short bursts but with different probabilities. The critical frequency (CF) for calcium electrogenesis in vitro was layer-specific and lower in L5 neurons compared to L2/3. Taking the in vitro CF as a predictive measure for dendritic electrogenesis during in vivo spontaneous activity, supracritical bursts in vivo were observed in a larger fraction of L5 neurons compared to L2/3 neurons but with similar incidence within these subpopulations. Together, these results show that in PFC of awake rats, AP spiking occurs at frequencies that are relevant for dendritic calcium electrogenesis and suggest that in awake rat PFC, dendritic calcium electrogenesis may be involved in neuronal computation.

Rajeevan T. Narayanan

Department of Integrative Neurophysiology,
Center for Neurogenomics and Cognitive Research,
Neuroscience Campus Amsterdam,
Department of Integrative Neurophysiology, Center for Neurogenomics and Cognitive Research, Neuroscience Campus Amsterdam

VU University Amsterdam

Juxtasomal Biocytin Labeling to Study the Structure-function Relationship of Individual Cortical Neurons