The brainstem, wedged between the spinal cord and the cortex, collects sensory input and distributes it throughout the brain, where it affects all manner of cognitive processing. For example, input from the brainstem’s locus coeruleus to the hippocampus shapes memory formation, and early pathology in the LC contributes to memory loss in Alzheimer’s disease. A study in the May 24 Science traced a new line of communication between the brainstem and the hippocampus, and showed that it, too, plays a critical role in memory formation. The work, from Gábor Nyiri and colleagues at the Hungarian Academy of Sciences, Budapest, reveals that neurons in the nucleus incertus connect to, and regulate the activity of, memory-storing CA1 interneurons in the hippocampus. Optogenetic activation of nucleus incertus GABAergic neurons prevented formation of fear memories in mice, while inhibition enhanced fear recall. The pathway may be involved in anxiety or stress disorders.

  • Brainstem’s nucleus incertus translates sensory input into GABAergic inhibition of hippocampal interneurons.
  • This helps set the strength and specificity of fear memories in mice.
  • Potential for role in dementia remains to be seen.

“This study adds more evidence to the fascinating role of evolutionarily ancient brainstem nuclei, such as the locus coeruleus and the nucleus incertus in higher-order cognitive functions,” wrote Heidi Jacobs, Maastricht University, The Netherlands, to Alzforum (full comment below).

Could NI dysfunction explain an aspect of dementia? There’s no direct evidence of that. Still, given the new data, “It is conceivable that dysfunction of NI GABAergic neurons may contribute to memory deficits as seen in Alzheimer’s disease,” according to Yadong Huang, University of California, San Francisco (full comment below).

NIght Light. Scheme for pairing foot shock with a specific environment to create context-dependent fear memories. The next day, control mice respond normally, with fear, in the same environment. Mice with light-activated NI neurons (ChR2 mice) do not. [Courtesy of Science/AAAS.]

The brainstem bears early signs of AD pathology. Phosphorylated tau protein appears in the locus coeruleus (LC) and is thought to spread from there (reviewed in Weinshenker 2018). Dysfunction and degeneration of LC neurons likely contributes to symptoms of AD. The same is true for Parkinson’s disease, where α-synuclein inclusions collect early on in the LC.

The nucleus incertus, on the other hand, has not been identified as a site of pathology, or linked to changes in brain connectivity or cognition in AD. Packed with GABAergic projection neurons, the NI modulates hippocampal theta rhythms, which are linked to learning and memory.

In the new study, first author András Szőnyi and colleagues drew on an arsenal of techniques, including tract tracing, immunogold receptor labeling and electrophysiological recording, to establish that NI GABAergic neurons directly and indirectly inhibit the firing of CA1 somatostatin-containing interneurons in the hippocampus. These interneurons, in turn, determine whether memory engrams are successfully encoded by pyramidal neurons. Environmental stimuli, especially noxious air puffs or rewarding sips of water, activated the NI neurons, suggesting the neurons might play a role in context-dependent memory formation, such as when mice learn to associate a painful shock with a specific locale.

To prove this, the scientists expressed light-responsive channels in GABAergic NI cells by way of optogenetics. They induced a contextual fear memory in mice by moving the animals from their home cage to a new environment, where the mice received a series of electric foot shocks. A day later, mice placed in the same environment froze in fear, anticipating a foot shock. If the researchers used light to activate NI neurons at the time of the foot shock, the mice did not make the fear memory and did not freeze in the new environment the next day. If the researchers inhibited NI activity, the mice seemed excessively scared, freezing for longer than control mice. This is consistent with what’s seen in NI-lesioned rats who display pathologically exaggerated fear memories (Pereira et al., 2013). 

The results suggest the NI acts via interneurons to fine-tune pyramidal cell recruitment, ensuring that the correct number of them form a memory. If the NI is overly active, too many pyramidal cells encode a memory and it does not stick, as in the fearless mice. When the NI is sluggish, too fewvpyramidal cells get involved and result is a stronger generalized fear, as in the NI-lesioned animals.

“The disruption of hippocampal theta and learning by targeted intervention in the brainstem GABAergic inputs is compelling. It will be interesting to know if other behaviors are affected,” said Steven Mennerick, Washington University in St. Louis.

The authors speculate that hyperactivity of NI might be linked to dementia, where memory formation is impaired. That has not been shown. “This is a nice study demonstrating NI GABAergic neurons bi-directionally regulate fear memory via somatostatin-positive interneurons in the hippocampus. Concerning a potential link to dementia, we will need to know if these neurons are dysregulated in dementia-related disorders,” said Li-Huei Tsai, MIT, Cambridge, Massachusetts.

Nyiri told Alzforum he does not believe that the function of NI has ever been tested in dementia or in any Alzheimer’s model. “But we know that prefrontal cortex is affected in Alzheimer’s disease, and we found that it projects onto NI GABAergic cells. Overactivity or degeneration of these inputs to the NI may have serious adverse consequences in memory formation, similar to that described in the paper,” he said.—Pat McCaffrey

Comments

  1. This study adds more evidence to the fascinating role of evolutionarily ancient brainstem nuclei, such as the locus coeruleus and the nucleus incertus in higher-order cognitive functions. The nucleus incertus consists of mostly GABAergic projection neurons, the main inhibitory neurotransmitter in the brain, and modulates hippocampal theta rhythms.

    The observation of Szonyi and colleagues that optogenetic stimulation of GABAerigc neurons in the nucleus incertus during learning was associated with reduced memory formation is consistent with previous work showing that the GABAergic agonist muscimol impairs memory, but the GABAergic anatagonist picrotoxin enhances memory (McGaugh, 1989). Interestingly, locus coeruleus hyperactivity has also been related to neurodegenerative diseases, as a greater turnover of norepinephrine was associated with increases in clinical symptoms (Weinshenker 2018; Sheline et al., 1998; Jacobs et al., 2019) and increases in tau burden (Jacobs et al., 2019).

    An intriguing question is how these associations between hyperactivity in brainstem nuclei and memory performance are connected to the pathogenesis of Alzheimer’s disease. Increased excitability of neurons has indeed been associated with tau accumulation (de Calignon et al., 2012), and therefore it is plausible to assume a link between both processes, though causality remains to be established.

    References:

    . Involvement of hormonal and neuromodulatory systems in the regulation of memory storage. Annu Rev Neurosci. 1989;12:255-87. PubMed.

    . Long Road to Ruin: Noradrenergic Dysfunction in Neurodegenerative Disease. Trends Neurosci. 2018 Apr;41(4):211-223. Epub 2018 Feb 20 PubMed.

    . Higher cerebrospinal fluid MHPG in subjects with dementia of the Alzheimer type. Relationship with cognitive dysfunction. Am J Geriatr Psychiatry. 1998 Spring;6(2):155-61. PubMed.

    . Alzheimer's disease pathology: pathways between central norepinephrine activity, memory, and neuropsychiatric symptoms. Mol Psychiatry. 2019 May 28; PubMed.

    . Propagation of tau pathology in a model of early Alzheimer's disease. Neuron. 2012 Feb 23;73(4):685-97. PubMed.

  2. This is a beautiful and exciting study showing conclusively that brainstem nucleus incertus (NI) GABAergic neurons control hippocampal-dependent contextual memory formation by inhibiting hippocampal somatostatin (SOM) interneurons in the stratum orients, both directly and indirectly through inhibition of excitatory neurons in the medial septum (MS). The study represents a great showcase of applying multidisciplinary approaches, including cell-type-specific neuronal tract tracing, immunogold receptor localization with electron microscope, electrophysiology, behavioral test, and optogenetic regulation, to complementarily address a key scientific question.

    The study suggests, as the authors concluded, that a role of NI GABAergic neurons may be fine-tuning of the selection of memory-encoding pyramidal cells in the CA1 of the hippocampus, on the basis of the relevance and/or modality of environmental inputs. The NI GABAergic neurons may also help filter non-relevant everyday experiences, such as those to which animals have already accommodated, by regulating the sparsity of memory-encoding CA1 pyramidal neurons. Thus, it is conceivable that dysfunction of NI GABAergic neurons may contribute to memory deficit, as seen Alzheimer’s disease.

    In this regard, we have previously shown that human ApoE4, the major genetic risk factor for late-onset Alzheimer’s disease, induces a SOM interneuron deficit in the hippocampus of mice in an age-dependent manner (Andrews-Zwilling et al., 2010; Leung et al., 2012) and in induced pluripotent stem cell (iPSC)-derived human neuron cultures (Wang et al., 2018). Importantly, the ApoE4-induced SOM interneuron deficit is also associated with alterations of the hippocampal network activity and correlate with spatial learning and memory impairments in aged mice (Gillespie et al., 2016; Andrews-Zwilling et al., 2010). Thus, dysfunction or loss of neurons along the NI GABAergic neuron-hippocampal SOM interneuron-axis (or the NI GABAergic neuron-MS excitatory neuron-hippocampal SOM interneuron-axis) may contribute to memory impairment in Alzheimer’s disease.

    Interestingly, as shown in this study, NI GABAergic neurons project to SOM interneurons in both the CA1 and the hilus of the dentate gyrus in mouse hippocampus. It would be worthwhile to also study the potential regulatory role of NI GABAergic neuron on hilar SOM interneurons and its implication in memory process and formation.

    References:

    . Apolipoprotein E4 causes age- and Tau-dependent impairment of GABAergic interneurons, leading to learning and memory deficits in mice. J Neurosci. 2010 Oct 13;30(41):13707-17. PubMed.

    . Apolipoprotein E4 Causes Age-Dependent Disruption of Slow Gamma Oscillations during Hippocampal Sharp-Wave Ripples. Neuron. 2016 May 18;90(4):740-51. Epub 2016 May 5 PubMed.

    . Apolipoprotein E4 causes age- and sex-dependent impairments of hilar GABAergic interneurons and learning and memory deficits in mice. PLoS One. 2012;7(12):e53569. PubMed.

    . Gain of toxic apolipoprotein E4 effects in human iPSC-derived neurons is ameliorated by a small-molecule structure corrector. Nat Med. 2018 May;24(5):647-657. Epub 2018 Apr 9 PubMed.

  3. Arousal depends on the activation of ascending neural pathways originating in the brainstem (pons and medulla). The nucleus incertus (NI) is a conserved area within the pontine periventricular region that consists of ascending GABAergic projection neurons and glutamatergic neurons. These GABAergic neurons disinhibit and synchronize the activity of their forebrain targets, promoting the rhythms typical of awake conscious states. Theta and gamma rhythms are among these oscillations, and these rhythms are thought to play a role in the encoding phase of learning and memory function.

    This paper effectively demonstrates that optogenetic stimulation of NI GABAergic neurons decreases hippocampal theta power in vivo. By contrast, optogenetic inhibition of NI GABAergic neurons during fear conditioning was shown by these researchers to enhance contextual memory formation. These findings demonstrate the importance of NI GABAergic neurons in hippocampus-dependent fear-memory formation implicated in many anxiety disorders such as phobias and PTSD. But this work sheds little light on the formation of episodic memories associated with less-stressful life events, such as place field formation during exploration of a novel environment or performance on a novel-object-recognition task following a delay, both of which may be less dependent upon activation of the NI.   

    The NI is also the primary site of Relaxin‐3 neurons.  It has previously been shown that corticotropin-releasing factor (CRF) depolarizes NI cells. The spontaneous firing of Relaxin-3 but not non- Relaxin‐3 neurons was shown by other investigators to be strongly modulated and phase-locked, with the initial ascending phase of hippocampal theta oscillations via postsynaptic CRFR1 in a long-lasting and non-desensitizing manner (Ma et al., 2013). NI is thus an important site for CRF modulation of hippocampal theta rhythm via effects on GABA/Relaxin‐3  transmission.

    There is a growing body of literature that suggests that AD neuropathology begins with dysregulation of function within the brainstem and that this, in turn, plays a role in the earliest stages of AD. The locus coeruleus (LC, also located in the pons) with its ascending adrenergic projections has also been implicated in AD.  Activation of the LC has been shown to suppresses feed-forward interneurons, and to enhance theta activity in rat dentate gyrus (Brown et al., 2005).  

    The LC is also one of the brain regions shown to be the most vulnerable to the occurrence of age-related tauopathy.  While it is known that dogs deprived of sleep develop tau pathology, and that stress can disturb sleep, it is nevertheless unclear to this reviewer how disruption of NI function may relate to dementia or AD neuropathology per se based on the data presented in this paper. 

    It will be important to elucidate the role of the NI in tauopathy using relevant animal models of aMCI or AD.  Increased tau phosphorylation and aggregation has been reported in the hippocampi of mice overexpressing corticotropin-releasing factor and so, dysregulation of function within the pons may eventually prove to be the missing link between neural circuit function and this specific aspect of AD neuropathology. 

    That said, there is also an emerging body of literature implicating stress and arousal networks in sleep dysregulation and a similarly impressive body of literature implicating sleep dysregulation in memory deficits and AD. More work will be needed to parse out the nature of these complex relationships.

    References:

    . Heterogeneous responses of nucleus incertus neurons to corticotrophin-releasing factor and coherent activity with hippocampal theta rhythm in the rat. J Physiol. 2013 Aug 15;591(16):3981-4001. Epub 2013 May 13 PubMed.

    . Locus ceruleus activation suppresses feedforward interneurons and reduces beta-gamma electroencephalogram frequencies while it enhances theta frequencies in rat dentate gyrus. J Neurosci. 2005 Feb 23;25(8):1985-91. PubMed.

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References

Paper Citations

  1. . Long Road to Ruin: Noradrenergic Dysfunction in Neurodegenerative Disease. Trends Neurosci. 2018 Apr;41(4):211-223. Epub 2018 Feb 20 PubMed.
  2. . Electrolytic lesion of the nucleus incertus retards extinction of auditory conditioned fear. Behav Brain Res. 2013 Jun 15;247:201-10. Epub 2013 Mar 26 PubMed.

Further Reading

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Primary Papers

  1. . Brainstem nucleus incertus controls contextual memory formation. Science. 2019 May 24;364(6442) PubMed.