Anterior thalamic nuclei neurons sustain memory

Abstract

A hippocampal-diencephalic-cortical network supports memory function. The anterior thalamic nuclei (ATN) form a key anatomical hub within this system. Consistent with this, injury to the mammillary body-ATN axis is associated with examples of clinical amnesia. However, there is only limited and indirect support that the output of ATN neurons actively enhances memory. Here, in rats, we first showed that mammillothalamic tract (MTT) lesions caused a persistent impairment in spatial working memory. MTT lesions also reduced rhythmic electrical activity across the memory system. Next, we introduced 8.5 Hz optogenetic theta-burst stimulation of the ATN glutamatergic neurons. The exogenously-triggered, regular pattern of stimulation produced an acute and substantial improvement of spatial working memory in rats with MTT lesions and enhanced rhythmic electrical activity. Neither behaviour nor rhythmic activity was affected by endogenous stimulation derived from the dorsal hippocampus. Analysis of immediate early gene activity, after the rats foraged for food in an open field, showed that exogenously-triggered ATN stimulation also increased Zif268 expression across memory-related structures. These findings provide clear evidence that increased ATN neuronal activity supports memory. They suggest that ATN-focused gene therapy may be feasible to counter clinical amnesia associated with dysfunction in the mammillary body-ATN axis.

Keywords: Anterior thalamic nuclei; Episodic memory; Extended hippocampal system; Glutamatergic; Immediate early gene; Lesions; Mammillothalamc tract; Optogenetics; Recovery of function; Spatial memory; Theta; Working memory.

Graphical abstract

Fig. 1

Temporal schematic of the key parts of the experiment. ATN = anterior thalamic nuclei; MTT = mammillothalamic tract. LV-CaMKII-ChR2-mCherry = lentiviral vector carrying channelrhodopsin construct; Zif268 = immediate early gene neuronal activity marker. Created with BioRender.com.

Fig. 2

MTT lesions and viral transduction of ATN neurons. MTT lesions produced persistent spatial working memory deficits compared to Sham lesions. A, Photomicrographs of Luxol blue (myelin-specific stain) combined with cresyl violet (Nissl stain) showing the intact mammillothalamic tract (MTT) in a Sham lesion rat (left), one rat with 100% MTT lesion on the left and 83% lesion on the right (middle), and an example (right) from the 11 rats with 100% bilateral MTT lesions (two other rats failed the inclusion criterion of 80% bilateral damage). B, Placement of bilateral viral vector infusions of channelrhodopsin and optrodes focused on the AV subregion of the ATN. Red fluorescence indicates mCherry-positive transduced glutamatergic ATN neurons, which are predominantly transduced in the medial AV subregion and lateral AM subregion. Blue cones depict a conservative estimate of the distribution of effective light from the optic fibres (yellow bars), which can be up to 1.3 mm below the fibre tip. C, Sagittal schematic of electrode placements in dorsal HPC (CA1; yellow) and PFC (green), and optrode implant in the dorsolateral ATN (blue). D, Example of transduced neurons in the AV: the middle panel shows mCherry only, at 40x; the far-right panel at 60x shows co-expression of CaMKIIα immunofluorescence of glutamatergic neurons (green) and mCherry (red) based on co-expression (orange/yellow). Nuclei are stained with DAPI (blue). E, The mCherry and CaMKIIα co-expression (example arrow-heads) was confirmed in prefrontal (top left panel), subicular (bottom left panel), and retrosplenial (top and bottom-right panels) terminal regions. F, The 12 opsin-MTT rats showed persistent impairment in spatial working memory in the 12-arm maze compared to 9 opsin-Sham rats. Pre-stim refers to performance prior to any optogenetic theta burst stimulation. Abbreviations: AD, anterodorsal thalamic nucleus; AM, anteromedial subregion of the ATN; ATN, anterior thalamic nuclei; AV, anteroventral subregion of the ATN; AVDM, anteroventral thalamic nucleus, dorsomedial subdivision; AVVL, anteroventral thalamic nucleus, ventrolateral subdivision; HPC, hippocampus; PFC, prefrontal cortex. Error bars = ±SEM. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Optogenetic activation of ATN neurons with exogenously-triggered regular blue light theta-burst stimulation (TBS) improves spatial memory in rats with MTT lesions. A, Mean spatial working memory errors for the two groups on individual days. Exogenously-triggered regular TBS with blue-light (B) ameliorated the spatial working memory impairment in the opsin-MTT group. B, Based on the averaged errors over the four days of each light-stimulation condition shown in A, spatial memory errors were significantly reduced by blue-light stimulation in 10 of 12 opsin-MTT rats (red squares = opsin-MTT; green dots = opsin-Sham). C, Improved spatial working memory in the opsin-MTT group with regular blue-light TBS was confirmed in the replication experiment. D, By contrast to exogenously-triggered regular blue-light TBS, the opsin-MTT group continued to make substantially more errors than the opsin-Sham group when endogenously-triggered blue-light TBS was used. Error bars = ±SEM. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Optogenetic activation of ATN neurons using exogenous, but not endogenous, TBS enhances neural oscillatory activity. Exogenously-triggered optogenetic TBS was applied for the data shown in A and B. A, Power spectral density (PSD) and coherence in the 6.8 Hz–10.3 Hz range for the aggregated electrophysiology for the last 8 correct choices for B and O days shown in Fig. 3A. B, PSD and coherence from the replication experiment (last 8 correct choices for B and O days shown in Fig. 3C). C, Endogenously-triggered blue-light TBS did not alter PSD in the ATN, HPC, or PFC in either group (last 8 correct choices for B and O days shown in Fig. 3D); the only effect on coherence was to reduce ATN-PFC coherence in the Sham group. Error bars = ±SEM. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Zif268 expression in memory-related structures is reduced by MTT lesions and increased by exogenously-triggered regular blue-light TBS. A, Seven brain regions were assessed after rats foraged for food in an open field when they received exogenously-triggered TBS using blue-light in the ATN in one hemisphere and orange-light in the contralateral ATN. B, Expression in the prefrontal cortex. C, Expression in CA1 and subiculum. D, Expression in the retrosplenial cortex (Ant = anterior; post = posterior; sup = superior layers; deep = deep layers). Other abbreviations: CA1, hippocampal CA1; Cg1, cingulate cortex 1; Cg2, cingulate cortex 2; PrL, prelimbic cortex; Rga, retrosplenial granular a cortex; Rgb, retrosplenial granular b cortex; Sub, dorsal subiculum. Values are relative to bregma. Error bars = ±SEM. # denotes differences between the sham and MTT groups following orange light; * denotes changes related to the blue light condition observed in both groups. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)