MIT 9.14 Class 2 Neuroanatomical techniques

Transcription

1 Brain Structure and its Origins Spring 2005 Massachusetts Institute of Technology Instructor: Professor Gerald Schneider A sketch of the central nervous system and its origins G. Schneider 2005 Part 1: Introduction MIT 9.14 Class 2 Neuroanatomical techniques

2 Primitive cellular mechanisms present in one-celled organisms and retained in the evolution of neurons Irritability and conduction Specializations of membrane for irritability Movement Secretion Parallel channels of information flow; integrative activity Endogenous activity

3 The need for integrative action in multi cellular organisms Problems that increase with greater size and complexity of the organism: How does one end influence the other end? How does one side coordinate with the other side? With multiple inputs and multiple outputs, how can conflicts be avoided (often, if not always!)? Hence, the evolution of interconnections among multiple subsystems of the nervous system.

4 How can such connections be studied? The methods of neuroanatomy (neuromorphology): Obtaining data for making sense of this lump of porridge. We can make much more sense of it when we use multiple methods to study the same brain. E.g., in addition we can use: Neurophysiology: electrical stimulation and recording Neurochemistry; neuropharmacology Behavioral studies in conjunction with brain studies In recent years, various imaging methods have also been used, with the advantage of being able to study the brains of humans, cetaceans and other animals without cutting them up. However, these methods are very limited for the study of pathways and connections in the CNS.

5 A look at neuroanatomical methods

6 Sectioning Figure by MIT OCW.

7 Cytoarchitecture: Using dyes to bind selectively in the tissue -- Example of stains for cell bodies Specimen slide removed due to copyright reasons. Ventromedial nucleus of hypothalamus (VMH) Rat brain, Coronal section Nissl stain (cell bodies)

8 Fiber architecture Example: visualizing a chemical that binds to myelin Figure removed due to copyright reasons. Myeloarchitecture of human midbrain

9 Chemoarchitecture, example: Acetylcholinesterase stain: Layers and patches in rat midbrain Specimen slide removed due to copyright reasons.

10 More chemoarchitecture: Histochemistry applied to comparative neuroanatomy of the forebrain PIGEON TELENCEPHALON Striatum SQUIRREL MONKEY TELENCEPHA Dorsal Ventricular Ridge Neocortex Striatum See Nauta & Feirtag, p.30, for another example Sketches of photos of acetylcholinesterase-stained sections of telencephelon of pigeon (above) and squirrel monkey (below). Figure by MIT OCW.

11 Immunohistochemistry, example: Opiate receptor localization in rat brain

12 Golgi Stain: Used by Ramon y Cajal to study connectivity of the brain and spinal cord (Courtesy of Nathaniel McMullen. Used with permission.)

13 Santiago Ramon y Cajal, drawing at his microscope

14 Golgi Method: axons in spinal cord (Ramon y Cajal) Figure removed due to copyright reasons. Please see: Cajal, S., and Ramón Y. Histology of the Nervous System of Man and Vertebrates. Translated from the French by Neely Swanson, and Larry W. Swanson. 2 vols. New York, NY: Oxford University Press, ISBN:

15 Ramon y Cajal: Neurons of spinal cord Figure removed due to copyright reasons. Please see: Cajal, S., and Ramón Y. Histology of the Nervous System of Man and Vertebrates. Translated from the French by Neely Swanson, and Larry W. Swanson. 2 vols. New York, NY: Oxford University Press, ISBN:

16 Ramon y Cajal: Diagram of local reflexes Cajal saw an entire S-R pathway for the first time. Figure removed due to copyright reasons. Please see: Cajal, S., and Ramón Y. Histology of the Nervous System of Man and Vertebrates. Translated from the French by Neely Swanson, and Larry W. Swanson. 2 vols. New York, NY: Oxford University Press, ISBN:

17 Brain connections and behavior The story of Karl Lashley s encounter, as a young student, with slides of a frog brain. If I could use this kind of material to see all of the connections, it would be possible to explain the frog s behavior. Assumption: the S-R model originating with René Descartes, championed by LaMettrie in the following century, boosted by the Russians Sechenov and Pavlov. Later, Lashley argued against the adequacy of S-R theory for explaining temporal order in rapid sequences of behavior Return to the primitive cellular mechanisms: the role of endogenous activity. (More about that later.) Motivational systems can initiate behavior, using inputs as guides. Nevertheless, the S-R model remains a common assumption among neuroscientists.

18 Connectivities: How do we know about them? Dissection Staining techniques: cells, fibers; uniqueness of Golgi methods Complexity problem: How to be sure of a connection? Historical example: How does info get from eye to neocortex? Electrophysiology: Sherrington et seq.; antidromic stimulation and recording Marchi method: an experimental anatomical technique. Nauta methods for silver staining of degenerating axons. Labeled amino acids and autoradiography HRP histochemistry: 2-way transport utilized Fluorescent tracers Immunohistochemistry: chemoarchitecture; new tracers, e.g. CT-B.

19 Walle J. H. Nauta From the Netherlands, then came to USA via Switzerland Father of modern experimental neuroanatomy M.I.T. Professor, (Institute Professor from 1973) First neuroanatomist to be appointed to the faculty of a psychology department (1964, MIT). This move by Hans-Lukas Teuber presaged the development of modern neuroscience.

20 Walle Nauta Photograph removed due to copyright reasons. M.I.T. Institute Professor Walle J. H. Nauta, M.D., Ph.D.

21 Example: Hamster with unilateral lesion of midbrain surface on first postnatal day: tracing of retinal projections from left eye, using a modified Nauta silver-stain for degenerating axons Photomicrograph(s) removed due to copyright reasons.

22 HRP staining, after anterograde transport from retina of hamster pup: labeled axons seen in diencephalon using dark-field microscopy Photomicrograph(s) removed due to copyright reasons.

23 Bright field Immunohistchemical staining for Cholera Toxin, subunit B (anterograde transport from part of retina to lateral geniculate nucleus) Photomicrograph(s) removed due to copyright reasons. Dark field

24 Retrograde tracer: Fluorogold (transport from SC to retina, seen in whole mount) Photomicrograph(s) removed due to copyright reasons.

25 Double Labeling: Nuclear Yellow and HRP (retrograde transport from optic tract to retina, seen in retinal whole mount) Photomicrograph(s) removed due to copyright reasons.

26 Co-localization: fluorogold & fluorescent beads

27 Primitive cellular mechanisms present in one-celled organisms and retained in the evolution of neurons Irritability and conduction Specializations of membrane for irritability Movement Secretion Parallel channels of information flow; integrative activity The need for neuroanatomical and other methods of research Endogenous activity

28 Endogenous activity: The primitive cellular mechanism often neglected Reasons for oversight: Forgetting about evolution The simplicity of the reflex model The discomfort of dealing with actions where the causes are not known. (Only with modern cell biological approaches are endogenous activities beginning to be understood.) Examples from neurons Example of hydra behavior

29 Example of endogenous activity in CNS (Spontaneous CNS activity) Endogenously generated rhythmic potentials in neuronal membranes can cause bursting patterns of action potentials Felix Strumwasser s Aplysia (sea slug) recordings (1960s, early 70s) There are many electrophysiological and molecular studies of endogenous activity in neurons since the early work. The biological clock: Control of circadian rhythms in vertebrates

30 Endogenous oscillator

31 Felix Strumwasser s Aplysia (sea slug) experiments Recordings form an identifiable large secretory neuron of the abdominal ganglion: T=40 sec (rhythm persists if action potentials are blocked with TTX), but not if sodium pump is blocked with Ouabain. This cell also showed a circadian rhythm that could be entrained by light.

32 Circadian rhythms in vertebrates Dependence on such biological clocks with a period of approximately 24 hr. Give mice heavy water, D 2 O, and their free-running circadian activity rhythm slows down to a degree proportional to the % D 2 O in their drinking water.

33 Selected References Slide 10: Slide 6: Figure by MIT OCW. MIT Based on: Striedter, Georg F. Principles of Brain Evolution. Sunderland, MA: Sinauer Associates, 2005, p.34. ISBN: Slide 6: Figure by MIT OCW. MIT 2006 Slide 13: Self Portrait of S. Ramon y Cajal, Slide 21: Image by Gerald Schnieder. G.E. Schneider 2006 Slide 22: Image by Gerald Schnieder. G.E. Schneider 2006 Slide 23: Image by Gerald Schnieder. G.E. Schneider 2006 Slide 24: Image by Gerald Schnieder. G.E. Schneider 2006 Slide 25: Image by Gerald Schnieder. G.E. Schneider 2006 Slide 26: Image by Gerald Schnieder. G.E. Schneider 2006 Slide 30: Drawing by Gerald Schnieder. G.E. Schneider 2006