Manipulating neural activity in physiologically classified neurons: triumphs and challenges

Understanding brain function requires knowing both how neural activity encodes information and how this activity generates appropriate responses. Electrophysiological, imaging and immediate early gene immunostaining studies have been instrumental in identifying and characterizing neurons that respond to different sensory stimuli, events and motor actions. Here we highlight approaches that have manipulated the activity of physiologically classified neurons to determine their role in the generation of behavioural responses. Previous experiments have often exploited the functional architecture observed in many cortical areas, where clusters of neurons share response properties. However, many brain structures do not exhibit such functional architecture. Instead, neurons with different response properties are anatomically intermingled. Emerging genetic approaches have enabled the identification and manipulation of neurons that respond to specific stimuli despite the lack of discernable anatomical organization. These approaches have advanced understanding of the circuits mediating sensory perception, learning and memory, and the generation of behavioural responses by providing causal evidence linking neural response properties to appropriate behavioural output. However, significant challenges remain for understanding cognitive processes that are probably mediated by neurons with more complex physiological response properties. Currently available strategies may prove inadequate for determining how activity in these neurons is causally related to cognitive behaviour.     

Convergence of visual,haltere, and prosternai inputs at neck motor neurons of Calliphora erythrocephala

Summary Neck muscles of Calliphora erythrocephala, situated in the anterior prothorax, are innervated on each side by 8 motor neurons arising in the brain (cervical nerve neurons, CN1–8) and at least 13 motor neurons arising in the prothoracic ganglion (anterior dorsal and frontal nerve neurons, ADN1,2 and FN1-11). Three prominent motor neurons (CN6 and FN1,2) are described in detail with special emphasis on their relationships with giant visual interneurons from the lobula plate, haltere interneurons, and primary afferents from the prosternal organs and halteres. These sensory organs detect head movement and body yaw, respectively. Neuronal relationships indicate that head movement is under multimodal sensory control that includes giant motion-sensitive neurons previously supposed to mediate the optomotor response in flying flies. The described pathways provide anatomical substrates for the control of optokinetic and yaw-incurred head movements that behavioural studies have shown must exist.     

Associative fear learning enhances sparse network coding in primary sensory cortex

Several models of associative learning predict that stimulus processing changes during association formation. How associative learning reconfigures neural circuits in primary sensory cortex to "learn" associative attributes of a stimulus remains unknown. Using 2-photon in vivo calcium imaging to measure responses of networks of neurons in primary somatosensory cortex, we discovered that associative fear learning, in which whisker stimulation is paired with foot shock, enhances sparse population coding and robustness of the conditional stimulus, yet decreases total network activity. Fewer cortical neurons responded to stimulation of the trained whisker than in controls, yet their response strength was enhanced. These responses were not observed in mice exposed to a nonassociative learning procedure. Our results define how the cortical representation of a sensory stimulus is shaped by associative fear learning. These changes are proposed to enhance efficient sensory processing after associative learning.     

Allergic inflammation-induced neuropeptide production in rapidly adapting afferent nerves in guinea pig airways

In the vagal-sensory system, neuropeptides such as substance P and calcitonin gene-related peptide (CGRP) are synthesized nearly exclusively in small-diameter nociceptive type C-fiber neurons. By definition, these neurons are designed to respond to noxious or tissue-damaging stimuli. A common feature of visceral inflammation is the elevation in production of sensory neuropeptides. Little is known, however, about the physiological characteristics of vagal sensory neurons induced by inflammation to produce substance P. In the present study, we show that allergic inflammation of guinea pig airways leads to the induction of substance P and CGRP production in large-diameter vagal sensory neurons. Electrophysiological and anatomical evidence reveals that the peripheral terminals of these neurons are low-threshold Adelta mechanosensors that are insensitive to nociceptive stimuli such as capsaicin and bradykinin. Thus inflammation causes a qualitative change in chemical coding of vagal primary afferent neurons. The results support the hypothesis that during an inflammatory reaction, sensory neuropeptide release from primary afferent nerve endings in the periphery and central nervous system does not require noxious or nociceptive stimuli but may also occur simply as a result of stimulation of low-threshold mechanosensors. This may contribute to the heightened reflex physiology and pain that often accompany inflammatory diseases.     

Tissue preparation and immunostaining of mouse sensory nerve fibers innervating skin and limb bones

Detection and primary processing of physical, chemical and thermal sensory stimuli by peripheral sensory nerve fibers is key to sensory perception in animals and humans. These peripheral sensory nerve fibers express a plethora of receptors and ion channel proteins which detect and initiate specific sensory stimuli. Methods are available to characterize the electrical properties of peripheral sensory nerve fibers innervating the skin, which can also be utilized to identify the functional expression of specific ion channel proteins in these fibers. However, similar electrophysiological methods are not available (and are also difficult to develop) for the detection of the functional expression of receptors and ion channel proteins in peripheral sensory nerve fibers innervating other visceral organs, including the most challenging tissues such as bone. Moreover, such electrophysiological methods cannot be utilized to determine the expression of non-excitable proteins in peripheral sensory nerve fibers. Therefore, immunostaining of peripheral/visceral tissue samples for sensory nerve fivers provides the best possible way to determine the expression of specific proteins of interest in these nerve fibers. So far, most of the protein expression studies in sensory neurons have utilized immunostaining procedures in sensory ganglia, where the information is limited to the expression of specific proteins in the cell body of specific types or subsets of sensory neurons. Here we report detailed methods/protocols for the preparation of peripheral/visceral tissue samples for immunostaining of peripheral sensory nerve fibers. We specifically detail methods for the preparation of skin or plantar punch biopsy and bone (femur) sections from mice for immunostaining of peripheral sensory nerve fibers. These methods are not only key to the qualitative determination of protein expression in peripheral sensory neurons, but also provide a quantitative assay method for determining changes in protein expression levels in specific types or subsets of sensory fibers, as well as for determining the morphological and/or anatomical changes in the number and density of sensory fibers during various pathological states. Further, these methods are not confined to the staining of only sensory nerve fibers, but can also be used for staining any types of nerve fibers in the skin, bones and other visceral tissue.     

A major role for intracortical circuits in the strength and tuning of odor-evoked excitation in olfactory cortex

In primary sensory cortices, there are two main sources of excitation: afferent sensory input relayed from the periphery and recurrent intracortical input. Untangling the functional roles of these two excitatory pathways is fundamental for understanding how cortical neurons process sensory stimuli. Odor representations in the primary olfactory (piriform) cortex depend on excitatory sensory afferents from the olfactory bulb. However, piriform cortex pyramidal cells also receive dense intracortical excitatory connections, and the relative contribution of these two pathways to odor responses is unclear. Using a combination of in vivo whole-cell voltage-clamp recording and selective synaptic silencing, we show that the recruitment of intracortical input, rather than olfactory bulb input, largely determines the strength of odor-evoked excitatory synaptic transmission in rat piriform cortical neurons. Furthermore, we find that intracortical synapses dominate odor-evoked excitatory transmission in broadly tuned neurons, whereas bulbar synapses dominate excitatory synaptic responses in more narrowly tuned neurons.     

Nerve growth factor regulates central terminals of primary sensory neurons

Transection of peripheral sensory axons results in transganglionic degenerative atrophy of central terminals of the affected primary sensory neurons. Nerve growth factor applied at the central stump of the transected nerve prevents or delays transganglionic degenerative atrophy. It is concluded that, under normal conditions, nerve growth factor taken up by receptors at peripheral sensory nerve endings and transported retrogradely to perikarya in dorsal root ganglia, regulates synthesis of neuroproteins destined for maintenance of central terminals of these neurons. Accordingly, transganglionic degenerative atrophy is the consequence of failure of nerve growth factor to reach perikarya of primary sensory neurons.     

Nonclassical neuronal communications

Examples from classical neuronal communications are discussed in the light of biochemical and anatomical data. These are the nonsynaptic axo-axonic interactions of the enkephalinergic neurons on nerve terminals of peptidergic primary sensory afferents and dopaminergic nigrostriatal fibers. Examples of dendrites as presynaptic sites are discussed in three very different situations, namely, the dopaminergic dendrites of the substantia nigra neurons, the gamma-aminobutyric acid--ergic dendrites involved in reciprocal dendro-dendritic synapses in the olfactory bulb, and the peripheral branches of the substance P-containing primary sensory neurons.     

Tension receptors on the apodemes of muscles in the walking legs of the crab,Cancer magister †

1. Mechanoreceptors monitoring tension in working muscles are described in the Decapoda Crustacea.

2. The receptors are associated with apodemes of muscles in the walking leg and are well‐developed in the extensor and flexor of the meropodite (Figures 1, 2).

3. The unbranched dendrites of the receptor neurones innervate the tissues surrounding the insertions of the muscle fibres (Figures 3, 4, 5(A)).

4. The receptors show spontaneous activity with the M‐C joint at resting position and this activity increases when the muscle is stretched by holding the joint at a different position (Figure 7).

5. Isometric tension increase in the muscle recruits sensory units (Figures 8, 10(A)) and increases the activity of units firing (Figure 9).

6. Apodeme receptors may be an entirely distinct input channel from chordotonal organs (Figure 10(B,C)). Joint movements produced by a standard muscle stimulus against increasing loads reveal very different responses (Figure 11).

7. Attempts to determine whether chordotonal organs (CP1, Figures 5(B), 6) monitor isometric muscle tension (Figure 12) suggest possible complexities in their dynamic responses.

8. Abbreviations used in this paper are FASN flexor apodeme sensory nerve, EASN extensor apodeme sensory nerve, BASN bender apodeme sensory nerve, and OASN opener apodeme sensory nerve.     

Suboesophageal neurons involved in head movements and feeding in locusts.

The projections of nerves 6 and 7 of the locust suboesophageal ganglion (SOG) were stained by axonal filling with cobalt chloride. Nerve 6 contains two motoneurons which innervate neck muscles 50 and 51. Sensory neurons innervating hairs on the dorso-occipital region of the head also enter the ganglion through nerve 6 and terminate in a small bilateral plexus. The projections of the head hairs in nerve 6 do not overlap the arborizations of the motoneurons or the neurons of nerve 7, but lie in the same area as descending sensory neurons from wind-sensitive hairs of the front of the head. One branch of nerve 7 (7B) contains two fibres which innervate the salivary gland. These 'salivary' neurons (labelled SN1 and SN2) have their cell bodies in the ganglion. The second branch, 7A, contains sensory neurons from the submentum of the labium, which form four sensory plexuses, two dorsal and two ventral. The sensory plexuses from the submentum have specific regions of overlap with the salivary neurons and with the neck muscle motoneurons. We interpret these as indicating a flow of information from labial receptors signalling head and mouthpart movement to neurons involved in salivation and head movement. We further postulate that the anatomical separation of the various sensory plexuses is indicative of functional localization within the ganglion.     

Mechanical sensitivity of the facial nerve fibers innervating the anterior palate of the puffer,Fugu pardalis,and their central projection to the primary taste center

Mechanical and chemical sensitivity of the palatine nerve, ramus palatinus facialis, innervating the anterior palate of the puffer, Fugu pardalis, and their central projection to the primary taste center were investigated. Application of horseradish peroxidase (HRP) to the central cut end of the palatine nerve resulted in retrogradely labeled neurons in the geniculate ganglion but no such neurons in the trigeminal ganglion, suggesting that the palatine nerve is represented only by the facial component. Tracing of the facial sensory root in serial histological sections of the brain stem suggested that the facial sensory nerve fibers project only to the visceral sensory column of the medulla. Peripheral recordings from the palatine nerve bundle showed that both mechanical and chemical stimuli caused marked responses. Mechanosensitive fibers were rather uniformly distributed in the nerve bundle. Intra-cranial recordings from the trigeminal and facial nerves at their respective roots revealed that tactile information produced in the anterior palate was carried by the facial nerve fibers. Elimination of the sea water current over the receptive field also caused a marked response in the palatine nerve bundle or facial nerve root while this did not cause any detectable responses in the trigeminal nerve root. Single fiber analyses of the mechanical responsiveness of the palatine nerve were performed by recording unit responses of 106 single fibers to mechanical stimuli (water flow), HCl (0.005 M), uridine-5'-monophosphate (UMP, 0.001 M), proline (0.01 M), CaCl2 (0.5 M), and NaSCN (0.5 M). All these fibers responded well to one of the above stimuli; however, most taste fibers did not respond well to the inorganic salts. The palatine fibers (n = 36), identified as mechanosensitive, never responded to any of the chemical stimuli, whereas chemosensitive fibers (n = 70) did not respond to mechanical stimuli at all. The chemosensitive units showed a high specificity to the above stimuli: they tended to respond selectively to hydrochloric acid, UMP, or proline. The responses of the mechanosensitive units consisted of phasic and tonic impulse trains and the sensitivity of the units varied considerably. The results reveal that the facial nerve fibers innervating the anterior palate of the puffer contain two kinds of afferent fibers, chemosensory and mechanosensory respectively, and suggest that the convergence of the tactile and gustatory information first occurs in the neurons of the primary gustatory center in the medulla.     

Convergence of mechanosensory inputs onto neuromodulatory serotonergic neurons in the leech

By the frequency-dependent release of serotonin, Retzius neurons in the leech modulate diverse behavioral responses of the animal. However, little is known about how their firing pattern is produced. Here we have analyzed the effects of mechanical stimulation of the skin and intracellular stimulation of mechanosensory neurons on the electrical activity of Retzius neurons. We recorded the electrical activity of neurons in ganglia attached to their corresponding skin segment by segmental nerve roots, or in isolated ganglia. Mechanosensory stimulation of the skin induced excitatory synaptic potentials (EPSPs) and action potentials in both Retzius neurons in a ganglion. The frequency and duration of responses depended on the strength and duration of the skin stimulation. Retzius cells responded after T and P cells, but before N cells, and their sustained responses correlated with the activity of P cells. Trains of five impulses at 10 Hz in every individual T, P, or N cell in isolated ganglia produced EPSPs and action potentials in Retzius neurons. Responses to T cell stimulation appeared after the first impulse. In contrast, the responses to P or N cell stimulation appeared after two or more presynaptic impulses and facilitated afterward. The polysynaptic nature of all the synaptic inputs was shown by blocking them with a high calcium/magnesium external solution. The rise time distribution of EPSPs produced by the different mechanosensory neurons suggested that several interneurons participate in this pathway. Our results suggest that sensory stimulation provides a mechanism for regulating serotonin-mediated modulation in the leech.     

The role of sensory input in motor neuron sprouting control

Primary sensory neurons project to motor neurons directly or through interneurons and affect their activity. In our previous paper we showed that intramuscular sprouting can be affected by changing the sensory synaptic input to motor neurons. In this work, motor axon sprouting within a peripheral nerve (extramuscular sprouting) was induced by nerve injury at such a distance from muscle so as not to allow nerve-muscle trophic interactions. Two different procedures were carried out: (1) sciatic nerve crush and (2) sciatic nerve crush with homosegmental ipsilateral L3-L5 dorsal rhizotomy. The number of regenerating motor axons innervating extensor digitorum longus muscle was determined by in vivo muscle tension recordings and an index of their individual conduction rate was obtained by in vitro intracellular recordings of excitatory postsynaptic end-plate potentials in muscle fibers. The main findings were: (1) there are more regenerated axons distally from the lesion than parent axons proximally to the lesion (sprouting at the lesion); (2) sprouting at the lesion was negatively affected by homosegmental ipsilateral dorsal rhizotomy; (3) the number of motor axons innervating extensor digitorum longus muscle extrafusal fibers counted proximally to the lesion increased following nerve injury and regeneration but this did not occur when sensory input was lost. A transient innervation of extrafusal fibers by &#110 motor neurons may explain the increase of motor axons counted proximally to the lesion.     

The role of sensory input in motor neuron sprouting control

Primary sensory neurons project to motor neurons directly or through interneurons and affect their activity. In our previous paper we showed that intramuscular sprouting can be affected by changing the sensory synaptic input to motor neurons. In this work, motor axon sprouting within a peripheral nerve (extramuscular sprouting) was induced by nerve injury at such a distance from muscle so as not to allow nerve-muscle trophic interactions. Two different procedures were carried out: (1) sciatic nerve crush and (2) sciatic nerve crush with homosegmental ipsilateral L3-L5 dorsal rhizotomy. The number of regenerating motor axons innervating extensor digitorum longus muscle was determined by in vivo muscle tension recordings and an index of their individual conduction rate was obtained by in vitro intracellular recordings of excitatory postsynaptic end-plate potentials in muscle fibers. The main findings were: (1) there are more regenerated axons distally from the lesion than parent axons proximally to the lesion (sprouting at the lesion); (2) sprouting at the lesion was negatively affected by homosegmental ipsilateral dorsal rhizotomy; (3) the number of motor axons innervating extensor digitorum longus muscle extrafusal fibers counted proximally to the lesion increased following nerve injury and regeneration but this did not occur when sensory input was lost. A transient innervation of extrafusal fibers by gamma motor neurons may explain the increase of motor axons counted proximally to the lesion.     

NGF receptor-mediated reduction in axonal NGF uptake and retrograde transport following sciatic nerve injury and during regeneration.

Injury to the rat sciatic nerve leads to the induction of nerve growth factor (NGF) receptors on the denervated Schwann cells and their disappearance on the regenerating axons of the axotomized, normally NGF-sensitive sensory and sympathetic neurons. This disappearance in the axonal expression and retrograde transport of NGF receptors is associated with a similarly dramatic reduction in the axonal uptake and retrograde transport of NGF following axotomy and during regeneration. In view of the massive NGF synthesis occurring in the injured nerve, these results suggest that, while sensory and sympathetic neurons are the primary targets of NGF in the normal peripheral nervous system, the denervated Schwann cells may become its primary target in the aftermath of nerve injury.     

Responses of bender apodeme tension receptors in the Dungeness crab,Cancer magister

Receptors monitoring tension are located on the apodemes of the bender (productor propoditis) muscle of walking legs and chelipeds of the Dungeness crab, Cancer magister. The bipolar neurons form a nerve, the bender apodeme sensory nerve (BASN), which joins the CP1 chordotonal organ nerve proximally, BASN units exhibit spontaneous activity at rest, and fire bursts of action potentials to rapid passive reduction of the carpopodite-propodite joint. Isometric contraction of the bender muscle results in BASN output that is directly related to force. Afferent activity ceases upon quick release of isometric tension, when tension drops to zero, and the unloaded muscle contracts isotonically.     

The systems analysis approach to mechanosensory coding

An important problem in neuroscience is to obtain quantitative knowledge of how information is represented, or encoded, in the signals that nerve cells process and transmit. Sensory receptors have provided important models for the study of neural coding because their inputs can often be relatively easily controlled and measured, while the resultant activity is recorded. A variety of engineering concepts have been successfully applied to physiological sciences, particularly those related to control of dynamic systems. Linear systems analysis was one of the earliest methods used to probe sensory coding, and measurements such as step responses and frequency responses have become standard tools for describing sensory functions. Modern systems analysis has evolved to provide accurate and efficient linear identification of encoding in sensory receptors that use either graded potentials or action potentials. It has also led to nonlinear systems analysis, the creation of parametric nonlinear models, and measures of information coding by sensory neurons. These methods promise to provide important new knowledge about sensory systems in the future, especially when complemented with parallel biophysical and molecular studies of sensory neurons. Mechanoreceptors provided some of the earliest preparations for the investigation of neural coding, and both the linear and nonlinear properties of wide variety of vertebrate and invertebrate mechanoreceptors continue to be explored. This article is part of a special issue on Neuronal Dynamics of Sensory Coding.     

Sound-driven synaptic inhibition in primary visual cortex

Multimodal objects and events activate many sensory cortical areas simultaneously. This is possibly reflected in reciprocal modulations of neuronal activity, even at the level of primary cortical areas. However, the synaptic character of these interareal interactions, and their impact on synaptic and behavioral sensory responses are unclear. Here, we found that activation of auditory cortex by a noise burst drove local GABAergic inhibition on supragranular pyramids of the mouse primary visual cortex, via cortico-cortical connections. This inhibition was generated by sound-driven excitation of a limited number of cells in infragranular visual cortical neurons. Consequently, visually driven synaptic and spike responses were reduced upon bimodal stimulation. Also, acoustic stimulation suppressed conditioned behavioral responses to a dim flash, an effect that was prevented by acute blockade of GABAergic transmission in visual cortex. Thus, auditory cortex activation by salient stimuli degrades potentially distracting sensory processing in visual cortex by recruiting local, translaminar, inhibitory circuits.     

Distribution,immunohistochemical characteristics and nerve pathways of primary sensory neurons supplying the porcine vas deferens

The present study investigated: (1) the distribution and chemical coding of primary sensory neurons supplying the vas deferens in juvenile pigs by the use of retrograde tracing combined with double-labelling immunofluorescence, (2) nerve pathways from dorsal root ganglia (DRG) to the vas deferens by means of denervation procedures involving transection of the hypogastric or pelvic nerve combined with a retrograde tracing method, and (3) possible interactions of the substance P (SP)/calcitonin gene-related peptide (CGRP)-immunoreactive varicose nerve fibres on vas deferens projecting neurons (VDPN) in the anterior pelvic ganglion (APG). The vast majority of VDPN were found mainly in the lumbar L2, L3 and sacral S2, S3 pairs of DRG and showed a clear ipsilaterally organized projection pattern. Immunohistochemistry revealed that most of these neurons contained SP and/or CGRP, occasionally coexpressed with galanin. Interestingly, pronounced differences in the expression of SP and/or CGRP were observed between the lumbar and sacral VDPN in that most of the lumbar but less than half of the sacral neurons stained for these peptides. Denervation experiments showed that the neurons located within the lumbar DRG project through the ipsilateral hypogastric nerve, whereas those found within the sacral DRG send their processes through the ipsilateral and contralateral pelvic nerve. In the nerve-lesioned animals, especially in those with the hypogastric nerve cut, a dramatic reduction in the number of SP and/or CGRP-containing nerve terminals surrounding the efferent VDPN within the APG was observed. This study has disclosed the distribution and, for the first time, chemical coding and nerve pathways of vas deferens-projecting primary sensory neurons in a mammalian species, the pig. The results obtained also provide some novel information about the possible morphological and functional relationship between vas deferens-projecting primary sensory and pelvic efferent nerve cells.     

Association of postganglionic sympathetic neurons with primary afferents in sympathetic-sensory co-cultures

Functional coupling between sympathetic postganglionic neurons and sensory neurons is thought to play an essential role in the pathogenesis of certain chronic pain syndromes following peripheral tissue and nerve injury. The mechanism(s) underlying this interaction are enigmatic. The relative anatomical inaccessibility of sympathetic and sensory neurons in vivo complicates study of their interrelationships. We have developed a system for long-term co-culturing of explants of sympathetic chain ganglia and dorsal root ganglia from newborn rats. Co-cultures were labelled for tyrosine hydroxylase-like immunoreactivity and studied at the light and electron microscopic levels. Explanted ganglia of both types survived well in co-culture. They maintained their tissue type-specific histological properties, including neuronal and glial morphology, and characteristic glial–neuronal associations. Moreover, neurons maintained their characteristic neurochemical identity, at least to the extent that sympathetic neurons continued to express tyrosine hydroxylase and dorsal root ganglion neurons did not. Sympathetic neurons emitted numerous outgrowing processes (axons) some of which came into association with sensory neurons in the explanted dorsal root ganglia. Some apparently specific sympathetic-sensory contacts were observed, suggesting that a functional interaction may develop between sympathetic axons and sensory neurons in vitro.